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Columbia  Slntoergitp 
mtijeCttponftrwgork 

College  of  -pfipsirians  anb  s&urgeons 
Htbrarp 


LABORATORY   STUDIES    IN    TROPICAL 
MEDICINE 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Open  Knowledge  Commons 


http://www.archive.org/details/laboratorystudieOOdani 


LABORATORY   STUDIES 


IN 


TROPICAL  MEDICINE 


C.    W.    DANIELS,    M.B.Camb.,  M.R.CP.Lond. 

Lecturer  on  Tropical  Diseases  at  the  London  Hospital ;  formerly  Director  of 
the  London  School  of  Tropical  Medicine ;  Director  of  the  Institute  for  Medical 
Research,  Federated  Malay  States;  Member  of  the  Royal  Society  Malaria 
Commission  in  India  and  Africa,  and  in  the  Medical  Service  of  the  Colonies  of 
Fiji   and  British  Guiana 

AND 

H.    B.    NEWHAM,    M.R.C.S.Eng.,  L.R.C.P.Lond., 
D.P.H.Camb.,  D.T.M.  &  H.Camb. 

Director,  formerly  Demonstrator  London  School  of  Tropical  Medicine 


THIRD     EDITION 

Thoroughly  revised,  with  many  new  and  additional  illustrations 

PHILADELPHIA 

P.     BLAKISTON'S     SON    &    CO 

1012,     WALNUT     STREET 

igil 


I'RINTF.I*    IN    ENGLAND. 


PREFACE  TO  THIRD   EDITION. 


Since  the  appearance  of  the  last  edition  of  this  book  many 
advances  have  been  made  in  the  study  of  tropical  diseases. 
Although  no  revolutionary  discovery  is  to  be  recorded,  still, 
new  facts  dealing  with  disease  and  its  propagation  have  been 
made  out  and  new  details  of  technique  have  been  elaborated. 

The  book  is  intended  essentially  to  be  a  practical  one,  and 
although  it  is  necessary  to  give  brief  descriptions  of  the  more 
important  protozoa,  helminths,  &c,  in  no  sense  is  it  to  be 
taken  as  a  complete  account  of  parasites  in  general. 

The  classification  of  parasites,  insects,  &c,  is  constantly 
changing  as  new  facts  regarding  them  come  to  light,  and 
no  authoritative  schemes  of  classification  is  given.  Those 
inserted  have  been  found  by  practice  to  be  useful,  and 
although  differing  in  many  details  from  those  adopted  by 
authorities  at  the  moment,  will,  the  authors  believe,  be 
found  of  utility  to  the  average  man. 

New  details  and  additions  have  been  made,  which  it  is 
trusted  will  serve  to  enhance  the  value  of  the  book  as  an 
aid  to  the  practical  worker  for  whom  it  is  intended. 

Our  best  thanks  are  due  to  Professor  Minchin  and  Messrs. 
Macmillan  for  kindly  permitting  us  to  make  use  of  the 
illustrations  showing  the  development  of  Gregarines  and 
Rhinosporidium. 

C.  W.  D. 

H.  B.  N. 

May,   191 1. 


PREFACE  TO   SECOND  EDITION 


Rapid  advance  has  been  made  in  all  branches  of  tropical 
medicine  since  the  first  edition  was  published.  In  this  edition 
these  advances  are  considered,  and  especially  information  as 
to  the  known  carriers  of  disease  has  been  added,  including 
ticks,  biting  flies  and  fleas. 

The  subjects  are  now  so  large  that  a  certain  amount  of 
systematic  classification  has  become  necessary,  and  tables 
are  therefore  included  showing,  in  brief,  the  zoological 
relationship  of  parasites  and  of  their  carriers. 

A  sub-division  of  the  chapters  has  been  made,  as  it  is  hoped 
from  time  to  time,  by  publication  of  the  further  advances 
made,  to  supplement  the  information  given  in  each  chapter. 

The  general  idea  remains  the  same,  the  book  is  intended  for 
the  lonely  worker  in  his  private  laboratory. 

We  are  indebted  to  many  friends  for  advice  and  assistance, 
particularly  in  the  revision  of  proofs,  and  especially  to  Dr. 
H.  B.  Newham,  and  A.  W.  Balch,  Surgeon,  U.S.  Navy. 

C.  W.  D. 

A.  T.  - 
September,  1907. 


PREFACE  TO  FIRST  EDITION. 


The  object  of  this  work  is  to  assist  practitioners  in  the 
Tropics  in  the  application  of  simple  laboratory  methods  to 
the  practice  of  medicine. 

The  writer  has  had  personal  experience  in  several  countries 
of  the  peculiar  difficulties  that  a  student  desirous  of  advancing 
the  knowledge  of  tropical  medicine,  or  of  practising  it  con- 
scientiously, will  meet,  and  the  plan  of  study  advocated  is 
the  outcome  of  this  experience. 

The  subjects  include  an  outline  of  animal  parasitology  and 
the  development  of  the  best  known  of  these  parasites.  The 
part  played  by  insects  in  spreading  disease  is  so  important 
that  it  is  necessary  to  have  a  sound  working  knowledge  of 
the  more  important  known  carriers  of  disease. 

Chapter  vii.  has  been  kindly  written  by  Mr.  F.  V.  Theobald 
for  this  book,  so  as  to  enable  the  student  to  differentiate  the 
more  important  genera  of  the  Diptera.  No  exhaustive  study 
of  any  one  subject  has  been  made,  but  it  is  hoped  that  suffi- 
cient information  is  given  to  enable  the  practitioner  to  com- 
mence the  effective  study  from  the  laboratory  point  of  view 
of  the  more  important  problems.  Simple  methods  are  selected 
as  far  as  possible,  and  those  recommended  are  in  the  main 
those  adopted  by  the  writer  for  teaching  purposes  at  the 
London  School  of  Tropical  Medicine,  and  can  be  relied  on 
as  applicable  to  the  circumstances. 

Few  references  are  given,  as  the  practitioner  in  the  Tropics 
has  rarely  access  to  a  library. 

I  am  much  indebted  to  Dr.  G.  C.  Low,  Medical  Superin- 
tendent at  the  London  School  of  Tropical  Medicine,  for 
valuable  assistance,  and  the  revision  of  the  proofs  has  also 
been  kindly  undertaken  by  him. 


CONTENTS. 


Chapter  I. 
The  Laboratory         I 

Chapter  II. 
Post-mortem  Examinations ...         ...         21 

Chapter  III. 
Blood 40 

Chapter  IV. 
Animal  Parasites  found  in  the  Blood        ...         ...         ...         ...         71 

Chapter  V. 
Parasites  found  in  the  Blood  of  Animals  ...         ...         ...         ...       100 

Chapter  VI. 
Parasites  found  in  Blood  Plasma ...       no 

Chapter  VII. 
Parasites  other  than  Protozoal  found  in  Human  Blood  ...       124 

Chapter  VIII. 
Certain  Properties  of  Blood  Plasma  and  Blood  Serum  ...       141 

Chapter  IX. 
Arthropoda — Insecta  154 

Chapter  X. 
Diptera  ...         ...         ...         ...         ...         ...         ...         ...       159 

Chapter  XI. 
Mosquitoes      ...         ...         ...         ...         ...         ...         ...         ...       200 

Chapter  XII. 
Dissection  of  Mosquitoes    ...         ...         ...       237 


CON!  EN  rS 


(   HAll  I  i:     XIII. 

Demonstration  of  Development  of  Parasites  in  Mosquitoes    ...      252 
Chapter  XIV. 

Eggs,  Larvae  and  Pupae  of  Mosquitoes     ...         ...         ...         ...       261 

Chat ikk  XV. 
Fleas,  Lice,  and  Bed-bugs 283 

Chapter  XVI, 

Arachnoidea — Ticks,  Mites,  Porocephalus.    Crustacea — Cyclops     296 

Chapter    XVII. 
Pigment  Deposits  and  Degenerations  in  Tissues  ...         ...       311 

Chapter  XVIII. 
Parasites  in  the  Tissues       ...         ...         ...         ...         ...         ...       321 

Chapter  XIX. 


Fasces 


Intestinal  Parasites 


Urine 


Bacteriology   ... 


Measurements 


Statistics 


Charier  XX. 


Chapter  XXL 


Chapter   XXII. 


Chapter   XXIII. 


Chapter  XXIV. 


33o 
345 
37i 
378 
436 
45" 


Appendix. 

Tables — Various  Staining  Methods,  etc. 
Instruments  and  Reagents  ... 

Index 


477 
482 

489 


LIST    OF    ILLUSTRATIONS. 


3- 

4- 

5- 

6- 

7- 
8- 

9- 
io- 
1 1- 
12- 

13- 
14- 

15- 

16- 


17- 
18- 
19- 

20- 
21- 
22- 

23- 

24" 

25- 
26— 

27— 
28— 


Automatic  Bunsen  Burner  for  Methylated  Spirit 

"  Primus  "  Paraffin  Lamp 

A  useful  Microscope  for  tropical  work 

Micrometer  Eye  pieces 

Micrometer  Eye  pieces 

Micrometer  Eye  pieces 

Koch's  Steam  Sterilizer 

Hot  Air  Sterilizer    ... 

Hearson's  Incubator,  working  with  Petroleum  Lamp 

Hot  Air  oven  for  paraffin 

Paraffin  Bath 

Block  for  moulding  paraffin   ... 

Cathcart's  Microtome,  with  spray  bellows 

Swift's  Freezing  Microtome  ... 

Parts  of  Swift's  Microtome    ... 

Cambridge   Rocking  Microtome,  new  pattern  for  cutting 

flat   sections,  with  large   articulating  apparatus  and 

one  razor 
Diagram  to  illustrate  the  making  of  a  wet  blood  film 
Braddon's  method  of  making  blood  films 
Crenated,  vacuolated  and  buckled  corpuscles    ... 
Method  of  making  dry  films  with  two  slides 
Method  of  making  dry  films  with  needle 
Method  of  making  dry  films  with  gutta  percha 
Method  of  making  dry  films  with  two  cover  glasses 
Leucocytes 
Myelocytes 
Wide-necked    stoppered    bottle   for    staining   and    fixin 

blood  films 
Schematic  view  of  the  asexual  and  sexual  phases  of  the 

malarial  parasite 
Parasites  in  capillaries 


4 
6 

14 
14 

'4 
17 
18 

19 
3* 

3i 
3i 
34 
35 


37 
42 
43 
45 
49 
49 
50 
5o 
54 
60 

74 

77 
86 


xii.  LIST  OF   ii.i.rsTkw  i  IONS 

PIG.  PAGE 

29 — Phases  in   the   asexual    and    sexual  development   of  the 

quartan  parasite  ...  ...  ...  •••        89 

30 — Phases   in  the   asexual   and  sexual   development  of  the 

benign  tertian  parasite    ...  ...  ...  •••         90 

31  —  Phases  in  the  asexual  and  sexual  development  of  the 
malignant  malarial  parasite 

32 — Proteosoma  and  Halteridium 

33 — Development  of  Piroplasma... 

34a — Drepanidium 

34^ — Hamiogregarina  balfouri 

35 — Development  of  II.  balfouri  (Plate)    ... 

35<z — Development  of  Gregarines... 

36 — A  Trypanosome  dividing 

37 — Forms  of  SJnrochtsta  obermeieri 

38 — Trypanosome  in  various  stages 

39 — Plate  of  Leishman-Donovan  bodies     ... 

40 — Trypanosomes  and  Leishman-Donovan  bodies  .  . 

41 — Method  of  making  a  film  for  the  examination  for  filarial 

42 — Glass  rack  for  staining  a  number  of  slides 

43 — Cobb's  formula  for  measuring  filariae  ... 

44 — Head  of  Filaria  bancrofti 

45 — Head  of  Filaria  ozsardi 

46 — Tail  of  Filaria  bancrofti 

47 — Tail  of  Filaria  ozzardi 

48 — Head  of  Filaria  demarquayi... 

49  —  H  ead  of  Filaria  perstans 

50 — Tail  of  Filaria  demarquayi  ... 

51 — Tail  of  Filaria  perstans 

52 — Some  of  the  important  Spectra  {Coloured  Plato 

53 — Wright's  Tubes 

54 —Mixing  Tube 

55 — Wright's  Tube  with  rubber  teat 

56 — Antennae   ... 

57 — Mouth  of  an  Empis 

58— Wing  of  Tipula 

59 — Base  of  wing,  calyptrate  diptera 

60— Puparium  of  a  "Screw-worm" 

61 — Wing  of  a  Cecidomyia 

62 — Wing  of  Anopheles  maculipennis 

63 — Wing  of  a  Culex 

64 — Wing  of  Chironomus 

65 — A  Ceratopogon 

66 — Wing  of  Ceratopogon  (after  Leonardi)... 

67 — Phlebotonuts 

68 — Wing  of  Simulium  ... 


LIST   OF   ILLUSTRATIONS 


69 — Head  of  Tabanus     ... 

70 — Wing  of  a  Tabanus... 

7 1  —  Tabanus  bo  vinus 

72 — Hcematopota  pluvialis 

73 — Head  of  Hcematopota 

74 — Wing  of  Hcematopota  pluvialis 

75 — Chrysops  dislinctipennis 

76 — Leptis  scolopacea 

77 — Wing  of  Empis 

77a— Wing  of  Syrphid;  wing  of  Phoridce... 

78 — Dermatobia  noxialis 

79 — Dermatobia  noxialis 

81 — Wing  of  Stomoxys  calcitrans. 

82 — Cross-section  of  proboscis  of  Stomoxys 

83 — Dissections  of  the  abdomen  of  Stomoxys 

84 — Transverse  section  of  the  proboscis  of  Glossina palpalis 

85 — Glossina  morsitans  ... 

86 — Lucilia  ccesar 

87 — Head  of  Lucilia  ccesar 

88 — Ch  rysomyia  macellaria 

89 — Aucluneromyia  luteola 

90 — Homalomyia  canicularis 

91 — Larva  of  Homalomyia 

92 — Wing  of  Hydrotcea  ciliata 

93 — Hippobosca  equina  . . . 

94 — Melophagus  ovinus. . . 

95 — Pinning  Mosquitoes  on  discs 

96 — Examination  of  scales  on  Mosquito     ... 

97 — Types  of  scales,  head  ornamentation,  forms  of  clypeus 

98 — Anatomy    ... 

99 — Types  of  metathorax  (Theobald) 
100 — Neuration  of  wing  (Theobald) 
101 — Various  forms  of  wing-scales  (Theobald) 
102 — Mosquitoes 

103 — Culex  (male  and  female)  and  Anopheles  (male  and  femal 
104 — Head  and  mouth-parts  of  Mosquito    ... 
105 — Maxillae  and  mandibles  of  Mosquito  ... 
106 — Tip  of  Proboscis  of  Mosquito 
107 — Cross-section  of  proboscis  of  Mosquito 
108 — Method  for  dissecting  Mosquitoes 
109 — Method  for  dissecting  Mosquitoes 
no — Method  for  the  dissection  of  the  salivary  glands 
1 1 1 — Method  for  the  dissection  of  the  salivary  glands 
112 — Internal  anatomy  of  the  Mosquito 
113 — Stomach  of  the  Mosquito,  showing  zygotes 


174 
174 
176 
176 

177 

177 
178 
179 
180 
181 
182 
182 
185 
186 
187 
189 
189 
192 
193 
193 
194 
196 
197 
197 
198 
198 
202 
203 
204 
206 
207 
209 
210 
214 
215 

237 
238 

239 
240 
242 

243 
245 
246 
248 

255 


XIV. 


LIST   <  IF   ILLUSTRATIONS 


'4 

'5 
16- 

17- 

t8 

19- 

20 

[21- 
12- 

23- 

-4- 

25- 

[26- 

[27- 

[28- 

:2a- 

30- 

Jt- 

32- 

t33~ 

134- 

'35- 

'36- 

137- 
138- 

'39- 
[40- 
141- 
142- 
143- 
'44- 
145- 
146- 
'47- 
[48- 

'49- 


'52- 
'53- 
'54- 

55- 
56- 


Eggs  of  Mosquitoes 

M 1  tin k1  for  cati  hing  larvae    ... 

Method  ii>r  cati  hing  larvae    ... 
-Head  of  Mosquito  larva 

Breeding  grounds  of  Mosquitoes 
-Breeding  grounds  <>i  Mosquitoes 

Mosquito  l)ox 
-Folding  Mosquito  cage 
-Mosquito  house 

-Mouth-parts  of  a  Flea  (after  Wagner) 
-External  anatomy  of  Flea 
-Types  of  Fle;is 
-Pediculus  vestimenti 
- 1  ^hthirius  inguinalis 
-Cimex  lectularius     ... 
-Legs  of  Ticks 
-Jxodina  (female) 
-Ixodina  (males) 
-Mouth-parts  of  Ornithodoros 
-Mouth-parts  of  Ixodes 
-Mouth-parts  of  Rhipiccphalus 
-  Ornithodoros  savignyi 
-Demodex  follicularum 
-Stages  of  Cyclops    ... 
-Coccidia  life-cycle    ... 
-Rhinosporidium  Kinealyi 
-Negri  bodies 
-Spectrum  of  Urobilin 
-Wire-gauze  strainer 
-Eggs  of  some  of  the  Intestinal  Worms 
-Anatomy  of  a  segment  of  a  Tapeworm 
-Genital  pores  of  some  of  the  Tapeworms 
-Anatomy  of  a  Fluke 
-Oxyuris  vermicularis  (male  and  female) 
-TrichocejShalus  dispar  (male  and  female) 
-Male  and  female  Ankylostomes 
-Head  and  tail  of  male  A.  duodenate ;    head 

male  N.  americanus 
-Scheme  of  development  of  Amoeba     ... 
-Lamblia  and  Trichomonas     ... 
-Balantidium  colt 
-Erlenmeyer's  Flask... 
-Petri's  Dish 
-Cornet's  Forceps 
-Durham's  Tubes 


and  tail 


LIST   OF    ILLUSTRATIONS 


XV. 


FIG. 

158 — Centrifuge... 

1159 — Aspergillus,  Penicillium,  and  Mucor 

160 — Yeasts 

161 — Thoma's  Fhemocytometer,  by  Zeiss 

162— Oliver's  Tintometer... 

163 — Govvers'  Haemoglobinometer 

164 — Von  Fleischl's  Hsemometer  ... 


408 
419 
421 
439 
448 
449 
450 


Seven  Statistical  Charts 


466,  467,  468,  469,  470,  471,  473 


Coloured  Plates. 

Plate     I.  5  figures  page  112 

Plate    II.  2  figures  page  120 

Plate  III.  Blood  Spectra  page  144 

Plate  IV.  27  figures         page  at  end 

Plate    V.  21  figures         page      „ 

Plate  VI.  23  figures         page      „ 


Studies  in  Laboratory  Work. 


CHAPTER   I. 


The  Laboratory.— In  few  places  in  the  Tropics  is 
there  any  institution  that  corresponds  to  the  British 
idea  of  a  laboratory.  Tap-water,  gas  and  electric  light 
usually  have  to  be  dispensed  with  and  substitutes 
employed.  The  isolated  worker  has  to  arrange  and 
make  his  own  laboratory,  either  in  the  house  or  attached 
to  a  hospital.     A  separate  building  will  rarely  be  available. 

The  first  essential  is  a  good  light,  and  if,  as  is  usual, 
work  is  done  by  daylight,  the  light  must  come  neither 
from  east  nor  west.  A  north  or  south  aspect  should  be 
chosen,  according  to  whether  the  worker  is  north  or 
south  of  the  line,  so  as  to  avoid  direct  sunlight. 

A  corner  of  a  verandah  can  be  made  into  a  good 
laboratory  by  placing  blinds  or  jalousies  on  two  sides, 
and  leaving  only  the  one  side,  that  facing  north  or  south, 
open.  The  side  from  which  the  light  is  received  should 
be  closed  in  with  a  window  if  possible,  to  prevent  the 
entrance  of  rain  and  dust. 

Another  important  consideration  is  wind,  and  with  the 
wind  the  amount  of  dust.  If  there  is  a  glass  window  this 
is  of  less  importance,  but  if  working  on  an  open  verandah, 
a  portion  of  the  verandah  sheltered  from  the  prevailing 
wind  must  be  selected,  even  if  this  choice  involves  the 
sacrifice  of  the  most  favourable  light. 

If  a  room  with  a  north  or  south  aspect  is  not  available 


llll     LABORATORY 

any  other  aspect  will  suffice,  provided  that  there  is  a  deep, 
low  verandah  outside  the  window. 

On  tin-  wall  of  the  laboratory  should  be  fixed  .1  number 
of  plain  wooden  shelves.  One  of  the  lower  of  these,  at  a 
convenient  height,  should  he  strong  and  broad  enough 
to  receive  heavy  weights.  On  this  shell'  may  be  kept 
mosquito  cages,  maturing  larva.'  and  other  objects 
awaiting  immediate  examination  or  requiring  constant 
attention. 

It  is  convenient  to  have  two  tables — one  on  which  to 
work  with  the  microscope,  and  also  tor  papers,  notebooks 
and  any  book  actually  in  use,  another  on  which  staining 
processes,  dissections  and  the  rougher  and  more  messy 
work  can  be  done.  Individual  habits  of  neatness  and 
arrangement  make  a  difference.  Though  much  excellent 
work  has  been  done  by  untidy  workers,  there  is  no  doubt 
that  in  the  limited  space  available  on  the  narrow  veran- 
dahs usual  in  many  parts  of  the  Tropics  work  is  easier 
and  more  comfortable  if  the  habit  of  tidiness  be  culti- 
vated. Persons  who  are  exceptionally  neat  and  method- 
ical in  their  habits  will  probably  find  one  long  table 
more  convenient  than  the  two  recommended  here. 

For  work  with  the  microscope  a  firm  steady  table  is 
required,  and  this  should  be  placed  a  lew  feet  from  the 
window.  The  second  table,  which  should  be  also  strong, 
must  be  placed  in  a  good  light,  and  it  is  better  to  have 
the  light  falling  from  the  left-hand  side  of  the  table. 

For  the  other  side  of  the  laboratory  jalousies  are  most 
convenient,  as  they  let  in  plenty  of  air  and  can  be  turned 
so  as  to  regulate  the  amount  of  air  and  to  stop  the 
entrance  of  rain.  A  cheaper  arrangement  is  to  use  reeds, 
as  natives  in  the  most  parts  of  the  world  are  good  workers 
with  reeds.  Sufficient  air  will  pass  through  to  keep  the 
room  cool.  Native  mats  can  be  used,  but  must  be  nailed 
on  to  a  framework,  otherwise  they  will  be  blown  about 
by  the  wind. 

Water  must  be  kept  in  bulk,  as  tap-water  is  rarely 
available.      A   small    tank — an   empty,  thoroughly   cleaned 


THE    LABORATORY 


kerosine  tin  will  serve — should  be  kept  filled  with  water. 
This  should  be  filtered,  and  a  glass  syphon  tube  with 
a  rubber  tube  and  clamp  attached  can  be   used  to  draw 


Fig.   i. — Automatic  Bunsen  Burner  for  Methylated  Spirit. 

it  off.  The  tank  must  be  kept  covered  with  a  well- 
fitting  lid,  and  a  basin  or  other  receptacle  should  be 
placed  underneath  to  receive  the  waste  and  washings  of 
stains,  &c. 


4  LABORATORY    AIMWkWl  I'S 

Distilled  water  must  be  kepi  in  bulk  in  a  well-stop- 
pered bottle,  from  which  a  sufficient  amount  is  taken  as 
required  into  a  wash  bottle  for  immediate  use. 

An  excellent  substitute  for  the  ordinary  gas  Bunsen 
burner  is  the  spirit  Bunsen  (fig.  i).  The  "Primus" 
Kerosine  Smokeless  Burner  will  be  found  very  useful  for 
heating  vessels  on  a  larger  scale  (fig.  2). 


Fig. 


Primus"  Paraffin  Lamp. 


An  incubator  is  an  enormous  advantage  and  tor  accurate 
bacteriological  work  is  essential.  The  temperature  in  most 
tropical  places  ranges  from  75'  upwards,  and  organism- 
grow  better  at  "  room  "  temperature  than  in  England.  In 
many  places  the  nocturnal  and  diurnal  variations  are 
small,  and  in  such  the  need  for  an  incubator  is  not  so 
great.  In  others  there  is  a  great  difference  between  the 
day  and  night  temperature,  and  in  these  the  need  is 
great.*  A  cold  incubator  is  useless  unless  ice  can  be 
obtained. 


::  With  practice  and  the  exercise  of  some  ingenuity  a  workable 
incubator  can  be  m;ide  by  placing  one  tin  inside  a  larger  one 
(or  a  chemist's  water-oven  may  be  employed).     The  space  between 


THE   MICROSCOPE  5 

Cultures  should  be  kept  in  a  dark  cupboard,  which 
must  he  as  dry  as  possible. 

Above  the  long  broad  shelf  running  along  the  wall 
two  or  three  rows  of  narrow  shelves  should  be  fitted  up 
on  which  stains  in  use  can  be  kept.  These  are  better  kept 
exposed  than  in  cupboards.  The  main  stock  can,  of 
course,  be  kept  out  of  sight. 

For  night  work  a  good  lamp  is  required.  The  lamp 
must  be  low,  and  the  flame  not  more  than  six  inches 
from  the  table.! 

Equipment  :  A  good  microscope  with  a  sub-stage  con- 
denser, iris  diaphragm  and  mechanical  stage  is  essential. 
An  oil  immersion  TV-inch  objective,  a  low  power,  say 
|-inch,  and  a  fairly  high  power,  say  £-inch,  will  be 
required.  For  many  purposes  a  i-inch  is  a  very  useful 
lens.     It  is  well  to  have  two  eye-pieces. 

The  choice  of  suitable  microscopes  is  a  large  one,  and 
the  differences  between  those  of  different  makers  are  not 
very  great,  the  points  of  difference  being  such  that  it  is 
difficult  to  say  which  is  the  best.  In  the  choice  much 
depends  on  the  conditions  under  which  the  work  has  to 
be  conducted.  If  much  travelling  has  to  be  done  it  is 
advisable  to  have  a  microscope  that  is  easily  carried  and 
can  be  set  up  for  use  at  a  moment's  notice.  Of  these 
travelling  or  portable  microscopes  there  are  several 
different  forms  all  fulfilling  the  main  requirements — light- 
ness, compactness,  and  usefulness.  The  folding  micro- 
scopes made  by  some  makers,  though  compact  and 
easily  packed,  are  heavy  and  therefore  inconvenient 
to  carry. 

If    most    of   the   work   can   be    done  at  a  fixed  station 


the  two  is  filled  with  water.  A  small  kerosine  lamp  placed  beneath 
the  tins  will  heat  the  water,  and  by  varying  the  height  of  the  lamp  a 
sufficiently  equable  temperature  can  be  maintained. 

t  For  the  best  definition  the  narrow  edge  of  the  flame  should  be 
used  as  the  source  of  the  illumination  and  focussed  accurately  on  the 
object. 


THE    .MK'k'OSCOl'K 


one  of  the  ordinary  forms  ol  microscope  is  the  mosl 
convenient  to  work  with.  It  the  expense  is  no  object 
it  is  well   to    have    two   stands,  one  portable  and  one  for 


Fig.  3. 

stationary  work.  The  objectives  and  eye-pieces  can  be 
used  for  either,  and  therefore  the  additional  expense  is 
not  very  great. 


THE    MICKOSCOI'K  7 

I 'arts  of  a  Microscope. — The  base  or  stand  is  a 
stage  fixed  either  to  a  tripod  or  to  a  vertical  column 
rigidly  attached  to  a  solid  and  heavy  footplate.  The 
tripod  is  to  be  preferred,  as  from  the  wide  spread  of 
the  legs  greater  stability  is  secured,  and  the  level  is  less 
affected  by  irregularities  in  the  table  on  which  the 
microscope  is  placed. 

In  the  folding  and  portable  microscope  the  legs  of  the 
tripod  are  jointed  at  or  near  their  junction  with  the  stage, 
and  can  be  folded  back  so  as  to  economize  space  in 
packing. 

The  stage  itself  is  a  fixed  plate  firmly  attached  to  the 
upright  carrying  the  optical  parts  of  the  instrument,  viz., 
the  mirror  and  sub-stage  condenser  below  the  stage,  and 
the  tube,  eye-piece  and  objective  above. 

To  this  solid  plate  is  fixed  the  mechanical  stage,  of 
which  there  are  two  main  types  : — 

(1)  Those  in  which  a  lighter  stage  carrying  the 
object  to  be  examined  is  attached  above  the  fixed 
stage.  This  can  be  moved  by  a  rack  and  pinion  in 
two  directions  at  right  angles  to  each  other. 

(2)  Those  in  which  the  slide  is  seized  by  catches 
and  moved  over  the  solid  stage. 

Some  mechanical  stages  have  in  addition  to  the  rectan- 
gular motions  a  circular  one  in  the  same  plane.  This 
motion  is  not  required. 

Of  the  two  types,  preference  should  be  given  to  the 
first,  as  it  can  be  used  for  objects  of  all  sizes  and  shapes, 
not  simply,  as  with  the  second,  for  objects  mounted  on 
the  regulation  slides.  With  care  it  does  not  get  out  of 
order  any  more  readily  than  that  of  the  second  tvpe. 

The  microscope  tube  is  attached  to  the  upright  in  such 
a  manner  that  it  can  be  moved  up  and  down  parallel  to 
the  upright,  but  allows  no  lateral  movement  in  any 
direction.  The  length  of  the  tube  is  important,  as  with 
the  higher  objectives  the  best  definition  is  obtained  with 
a  certain  known  length  of  tube.  This  distance  varies 
with  the  objectives  of  different  makers.     To  provide  for 


8  THE    MICROS!  OPE 

this  variation  there  i>  a  second  or  draw-tube  inside  the 
outer  tube,  which  can  be  drawn  out  so  as  to  lengthen  the 
tube  to  the  required  extent.  The  length  of  tube  required 
for  an  objective  should  be  ascertained,  and  the  draw-tube 
should  be,  and  usually  is,  marked  so  that  the  correspond- 
ing length  can  be  obtained. 

In  the  portable  and  folding  microscope  the  outer  tube 
is  so  short  that  it  is  always  necessary  to  use  the  draw  tube. 
The  adjustments  by  which   the  object    is  focussed  are 
of  two  kinds  : — 

(i)  The  coarse  adjustment,  by  which  the  tube  is 
moved  by  a  rack  and  pinion  and  brought  approxi- 
mately into  focus.  The  range  of  the  coarse  adjust- 
ment is  great,  but  the  movement  is  too  coarse  to 
focus  easily  and  correctlv  with  higher  powers. 

(2)  The  line  adjustment,  which  may  be  a  differen- 
tial screw  or  of  the  lever  pattern.  The  range  of  this 
adjustment  is  small,  but  very  delicate  movement  is 
obtained. 

Illuminating  Apparatus. — Good  illumination  is  abso- 
lutely necessary  for  useful  work  with  higher  powers.  The 
parts  of  the  microscope  providing  for  this  illumination 
and  modifying  it  are  the  mirror,  the  sub-stage  condenser 
and  the  iris  diaphragm,  where,  as  is  most  usually  the  case, 
the  object  is  to  be  examined  by  transmitted  light.  For 
opaque  objects  which  can  only  be  usefully  examined  with 
low  powers  illumination  comes  from  above  the  stage. 

The  Mirror  is  attached  below  the  condenser.  It  has 
two  surfaces,  one  concave  and  the  other  plane.  The 
plane  mirror  is  that  employed  for  work  with  higher 
powers.     Too  small  a  mirror  should   not  be  used. 

The  Sub-stage  Condenser. — This  is  placed  between  the 
mirror  and  the  stage,  and  collects  the  rays  of  light 
received  from  the  mirror  into  a  cone  of  large  aperture, 
which  can  be  focussed  on  to  the  plane  of  the  object. 

It  must  be  centred  so  that  the  optical  axis  corresponds 
with   that   of  the  objective,  and   must   be   movable  so  that 


THE   MICROSCOPE  9 

it  can  be  moved  up  or  down  in  this  axis.  The  move- 
ment is  better  performed  by  a  rack  and  pinion,  but  in 
most  of  the  portable  microscopes  this  has  to  be  done  by 
hand. 

The  Lenses. — To  the  tube  are  fixed  at  each  end  the 
two  systems  of  lenses  used  for  the  magnification  of  the 
object.  The  lower  system  of  lenses,  which  is  screwed 
on  to  the  lower  end  of  the  tube,  is  the  objective,  and 
forms  a  real  image  of  the  object,  which  is  further  magni- 
fied by  the  system  of  lenses  at  the  upper  end  of  the  tube 
— the  eye-piece. 

To  save  time,  annoyance,  and  wear  of  screws,  a  nose- 
piece  is  fitted  to  the  lower  end  of  the  tube,  to  which 
can  be  screwed  the  three  objectives  in  use  instead  of 
screwing  them  directly  to  the  lower  end  of  the  tube. 

These  are  the  essentials  of  a  microscope  for  the  work 
here  contemplated.  It  can  be  purchased  complete  for 
about  ^20  from  several  well-known  makers.  The  price 
varies  a  little,  but  the  reader  is  strongly  advised  to  pay 
little  attention  to  slight  differences  of  price  in  the  selec- 
tion of  an  instrument  that  suits  him.  Much  more  expen- 
sive instruments  can  be  purchased,  but  at  about  the 
above-mentioned  price  an  instrument  can  be  obtained 
suitable  for  the  work  contemplated.  The  portable 
microscopes  with  the  same  objectives  are  about  £3  or 
.£4  less. 

No  microscope  should  be  bought  without  spending 
some  time  in  careful  examination  and  testing  of  the 
lenses  and  adjustments.  The  points  to  which  special 
attention  should  be  paid  are  :  (1)  The  rigidity  of  the 
stand.  This  rigidity  must  be  constant  both  with  the  tube 
vertical  and  inclined.  (2)  All  the  adjustments  and  screw 
movements  must  be  tested  to  see  that  they  work  smoothly 
and  evenly,  and  that  every  movement  of  the  milled  head 
results  in  effective  movement  of  the  screw  and  of  the 
part  of  the  instrument  which  it  is  intended  to  move. 


io  i  UK   MICROSCOPE 

With  the  mechanical  stage  il  is  further  necessary  to 
satisfy  one's  self  that  the  movement  imparted  to  the  stage 
is  all  in  one  plane,  otherwise  as  the  object  is  moved  it  will 
also  move  out  of  focus.  This  can  be  ascertained  by 
examining  an  object,  such  as  a  uniform  blood-film,  under 
various  powers,  and  determining  how  far  the  object 
remains  in  focus.  When  using  a  y^-inch  objective,  even 
with  the  best  stages,  some  focussing  will  be  necessary, 
but  it  should  be  slight,  and  the  object  should  be  very 
little  out  of  focus  with  considerable  movements  of  the 
mechanical  stage.  A  slide  and  film  ot  uniform  thickness 
must  be  used  for  this  test,  and  the  result  of  the  examina- 
tion should  be  confirmed  by  using  a  series  of  slides. 

The  nose-piece  should  centralize  the  objective  cor- 
rectly, otherwise  an  object  that  is  in  the  centre  of  the 
field  with  a  low  power  may  not  be  in  the  field  with  a 
higher  power.  This  is  tested  by  centralizing  with  the 
highest  power  some  object  that  is  visible  with  the  lowest 
power,  and  seeing  how  near  the  centre  of  the  field  this 
object  is  when  viewed  with  the  other  objectives.  The 
order  should  also  be  reversed  if  the  test  appears  to  be 
satisfactory. 

In  testing  the  objectives,  the  points  to  be  most  closely 
investigated  are  : — 

(i)  Definition.  Unless  the  object  is  sharply  and 
clearly  defined,  the  magnification  is  wasted. 

(2)  Flatness  of  field.  Many  lenses  give  good  and 
sharp  definition  at  the  centre  of  the  field,  whilst 
objects  a  little  removed  from  the  centre  are  blurred, 
and  those  at  the  periphery  are  out  of  focus.  In 
using  such  lenses,  if  any  other  part  of  the  field  is 
brought  into  focus,  the  objects  in  the  centre  of  the 
field  will  be  out  of  focus.  With  such  a  lens  the  field 
is  not  fiat.  It  is  perhaps  too  much  to  hope  that  the 
periphery  of  the  field  will  be  in  sharp  focus  at  the 
same  time  a-  the  centre,  but  at  any  rate  for  blood 
work  the  greater  part  of  the  field  must  be  flat,  other- 
wise objects  such  as  malaria  parasites  may  easily  be 
overlooked. 


ILLUMINATION  II 

(3)  Chromatic  aberration  must  be  entirely  corrected 

and  no  particoloured  fringe  seen  round  the  edge  of 
the  field. 

(4)  Magnification.  As  a  test  object  a  well-stained, 
evenly  spread  blood-film  is  as  good  an  object  as  any, 
and  as  the  objeet  is  a  familiar  one  the  degree  of 
magnification  can  be  readily  estimated.  Both  eye- 
pieces should  be  used  in  turn. 

In  the  use  of  the  microscope  great  attention  must  be 
paid  to  the  illumination.  The  light  in  the  Tropics  is  not 
good,  as  it  so  often  has  to  be  derived  from  blue  skw 
The  mirror  should  be  turned  so  as  to  receive  the  light 
from  a  white  cloud  when  possible. 

When  very  sharp  definition  is  required  the  narrow 
edge  of  a  flat  flame  should  be  used  as  the  source  of 
illumination.  In  using  the  low  power  this  should  be 
focussed  on  the  object,  and  by  means  of  the  centring 
screws  of  the  condenser  brought  right  across  the  field  of 
vision.  A  higher  power,  say  1,  should  then  be  used,  and 
again  the  condenser  should  be  centred  so  that  the  image 
of  the  flame  stretches  across  the  field.  Finally,  this  pro- 
cess is  repeated  with  the  oil  immersion  lens. 

The  condenser  must  be  raised  until  the  image  of  the 
flame  is  as  sharply  defined  as  the  object  under  examina- 
tion. In  this  way  very  sharp  definition  is  obtained,  but 
the  greater  part  of  the  field  is  not  illuminated. 

For  the  finer  work  the  condenser  as  well  as  the 
objectives  must  be  apochromatic. 

In  using  a  low  power  the  condenser  should  be  low  so 
as  to  be  out  of  focus,  or  if  the  stand  permits  it,  swung 
out  so  as  not  to  be  between  the  mirror  and  the  object. 
With  a  J-inch  objective  it  should  be  higher,  and  with 
the  xV-inch  oil  immersion  objective  close  to  the  under- 
surface  of  the  slide. 

The  brightest  and  most  uniform  light  that  can  be 
obtained  with  the  iris  diaphragm  open  is  the  best.  If  it 
is  desired  to  reduce  the  light,  it  should  be  done  by  closing 
the  diaphragm,  not  by  altering  the  position  of  the  con- 
denser or  of  the  mirror. 


l  2  USE   OF   MICR4  >SC<  >PE 

Both  tlir  mirror  and  condenser  should  be  kept  clean. 
It    is  well   to  haw  a  spare  mirror,  as   these   silvered 
mirrors  sometimes  deteriorate  rapidly  in  the  Tropics. 
In   focussing  it   is  well  to  bring   the  objective  nearer 

to  the  object  than  is  necessary,  and  then,  using  the 
coarse  adjustment,  whilst  looking  down  the  microscope 
to  withdraw  the  objective  from  the  object  till  it  is  seen 
more  or  less  distinctly.  For  exact  focussing  the  line 
adjustment  should  be  used,  but  not  till  the  object  is 
nearly  in  focus.  The  range  of  the  tine  adjustment  is 
small,  and  if  used  over  too  extensive  a  range  there  is  risk 
of  straining  it. 

When  working  with  the  oil  immersion  lens  it  is  well 
to  place  the  oil  on  the  object  and  screw  down  the  tube 
till  the  objective  touches  the  oil.  In  doing  this  the  drop 
of  oil  should  be  viewed  from  the  side,  and  it  will  then 
be  easy  to  see  when  the  objective  touches  the  oil.  After- 
wards very  slowlv  focus  on  the  object.  Before  using  an 
oil  immersion  lens  the  held  should  be  examined  with 
a  low  power  to  make  certain  that  there  is  something 
visible  in  the  held.  In  a  fresh  blood-film,  for  instance, 
if  a  part  be  selected  in  which  there  are  no  corpuscles 
there  may  be  nothing  to  focus  on,  and  in  such  a  case 
there  is  risk  of  screwing  the  objective  down  on  the  cover- 
glass. 

If  black  specks  are  visible  in  the  field  it  is  well  to  rotate 
the  eye-piece;  if  these  rotate  with  the  eye-piece  there  are 
particles  of  dirt  in  some  part  of  the  eye-piece.  Dirt  on 
the  objective  shows  as  a  general  haziness;  such  haziness 
may  also  be  due  to  a  cloudy  or  dirty  cover-glass  or  a 
badly-prepared  specimen. 

All  glass,  and  particularly  the  softer  and  more  highly 
retractile  glass  of  which  lenses  are  made,  is  liable  in  a  hot, 
moist  climate  to  deteriorate  and  become  cloudy  or  white, 
resembling  very  line  ground  glass.  When  lenses  become 
affected  in  this  way  they  require  regrinding.  Some  lenses 
spoil  more  quickly  than  others,  and  in  purchasing  objec- 
tives it  should  be  stated   that  they  are  required  for  work 


DETERIORATION  1 3 

in  the  Tropics.  Various  less  serious  conditions  are  some- 
times mistaken  for  this  change  in  the  glass.  The  cement 
may  run  so  that  it  partly  covers  the  inner  aspect  of  the 
objective.  In  other  cases  water  condenses  between  two 
lenses  and  causes  a  want  of  definition  similar  to  that  due 
to  frosting  of  the  glass.  Either  of  these  conditions  may 
be  detected  by  unscrewing  the  lenses  and  examining  the 
surface  with  a  watchmaker's  glass  or  hand-lens.  These 
conditions,  when  discovered,  are  easily  remedied.  It  is 
well  to  use  only  lenses  that  can  be  unscrewed,  and  from 
time  to  time  to  unscrew  and  clean  the  surface  of  the 
lenses  carefully.  They  will  keep  longer  if  this  is  done, 
but  must  not  be  expected  to  last  as  long  as  they  do  in 
England.  Lenses  not  in  use  are  best  kept  in  a  perfectly 
dry  stoppered  bottle.  There  is  no  objection  to  having 
some  dehydrating  agent,  such  as  well-dried  calcium 
chloride,  in  a  separate  compartment  in  the  same  bottle. 

Lenses  after  use  are  best  cleaned  with  a  soft  rag  dipped 
in  alcohol  or  xylol.  If  these  are  not  at  hand  a  soft 
handkerchief  moistened  with  saliva  forms  an  excellent 
substitute. 

A  camera  lucida  or  drawing  camera  is  a  great  conve- 
nience, and  so  useful  for  measurements  that  some  form 
of  this  instrument  should  be  obtained.  That  of  Leitz  is 
a  cheap  and  simple  iform,  the  use  of  which  it  is  easy  to 
learn. 

For  measurements  a  micrometer  slide  ruled  to  y^o  of 
a  millimetre  is  a  useful  accessory  ;  failing  it,  any  of  the 
standard  ruled  scales,  such  as  the  counting  chamber  of 
a  Thoma-Zeiss'  or  Gowers'  hsemocytometer,  can  be  used 
as  a  substitute. 

A  micrometer  scale  (fig.  4),  to  be  placed  in  the  eye- 
piece in  focus  with  the  front  lens,  is  useful  for  some 
measurements,  but  can  be  dispensed  with  if  measure- 
ments are  made  with  a  camera  lucida.  A  more  useful 
form  of  eye-piece  micrometer  is  ruled  in  squares  (fig.  0.) 
Once  they  are  standardized  these  can  be  used  for  blood 
counts,    and    the   ruled   scales    used    for    the    counting 


14 


MICROMETER    EYE-PIEl  I 


chamber  of  ;i  haemocytometer,  &c,  dispensed  with.     For 
many  purposes  it   is  convenient   to  subdivide    the  field, 


Fro.  4. 


Fig.  5. 


^rfF-- 

-:=Fr>s. 

^L* 

vv 

z. 

\ 

z. 

/L 

1 

/_ 

-\ 

\ 

L~ 

1 

L                 

____} 

I           -   -     - 

17 

X  - 

X   7 

.    _      _   _       ' 

\~ 

X  ~         ::: 

17 

\    .  ::::: 

\            : 

^,         - 

--    -    " '  ^ 

^,      - 

jf 

Fig.  6. 


and  this   can    be  more    readily  clone  with  a  micrometer 
eye-piece  ruled  in  squares  than  in  any  other  way. 

These   eve-piece  scales   are   -imply    placed    in    the  eye- 


DISSECTING    MICROSCOPE  I  5 

piece  and  rest  on  the  diaphragm  between  the  two  lenses. 
It  will  usually  be  necessary  to  move  the  diaphragm 
slightly  in  order  to  bring  the  scale  sharply  into  focus,  but 
this  is  easily  accomplished. 

These  eye-pieces  require  standardization  tor  the  value 
of  the  squares  or  scale.  The  micro-millimetre  scale  is 
used  as  the  object,  and  for  each  objective  the  number 
of  micro-millimetres  in  a  division  of  the  scale  noted. 
This  can  be  done  once  for  all  and  the  records  preserved. 
There  is  no  object  in  having  the  divisions  of  a  scale  or 
the  squares  of  an  accurately  known  size.  As  seen  in  the 
eye-piece  they  are  magnified. 

A  warm  stage  is  not  so  much  needed  in  the  Tropics  as 
in  England,  but  is  a  convenience.  The  simplest  form  is 
a  copper  plate  perforated  with  a  hole  the  size  of  a  shilling. 
From  the  plate  a  copper  tongue  extends  in  front  for 
for  about  six  inches.  The  under-surface  of  the  plate  is 
covered  with  cloth  and  is  placed  on  the  stage  so  that  the 
aperture  corresponds  to  the  central  aperture  in  the  stage. 
The  object  is  placed  on  the  slide  on  the  copper  plate  and 
examined,  and  by  heating  the  tip  of  the  tongue  of  copper 
projecting  from  the  plate  by  means  of  a  spirit  lamp,  the 
heat  will  be  conducted  to  the  plate  and  the  slide  kept 
warm.  By  heating  the  tongue  nearer  to  the  plate  a  higher 
temperature  will  be  obtained,  and  by  lowering  the  spirit 
lamp,  or  moving  it  further  off,  a  lower  temperature. 
With  a  little  practice  there  is  no  difficulty  in  maintaining 
a  fairly  uniform  temperature  which  can  be  estimated  by 
touch.  More  elaborate  warm  stages  are  to  be  procured 
in  which  the  temperature  is  kept  steady  by  the  circula- 
tion of  hot  water. 

A  dissecting  microscope  is  useful  but  not  essential  ;  it 
consists  of  a  single  compound  lens  fixed  on  a  vertical 
carrier  which  can  be  raised  or  lowered  by  a  rack  and 
pinion ;  the  stage  is  of  glass  and  there  are  wooden 
movable  hand-rests  at  each  side.  For  illumination  there 
is  a  plane  reflector,  and  as  an  alternative  on  the  other 
side  of  the  mirror  a  plaster  of  Paris  disc. 


id  COVER   GLASSES   AND   SLIDES 

For  most  of  the  purposes  for  which  the  dissecting 
microscope  is  used  a  watchmaker's  glass  does  equally 
well,  and  for  some  purposes  it  is  better,  as  both  hands  arc- 
true,  and  no  stage  is  required. 

A  good  large  hand-lens  on  a  handle  is  useful  for 
observing  the  habits  of  mosquito  larvae. 

Reagents,  stains,  slides  and  cover-glasses  are  required. 
Tile  slides  should  not  be  of  the  best  quality  ;  the  thin, 
white  slides  deteriorate  in  the  Tropics  more  rapidly  than 
the  coarser  glasses.  No.  2  quality  is  to  be  preferred. 
They  require  thorough  cleaning,  and  a  stock  cleaned  and 
ready  for  use,  requiring  only  to  be  wiped,  should  be 
kept  in  hand.  They  arc  best  cleaned  by  placing  in  a 
saturated  solution  of  carbonate  of  soda  which  is  just 
brought  to  the  boil.  Afterwards  they  are  well  washed  in 
running  water,  wiped  with  a  soft  linen  rag,  and  kept  in 
spirit  in  a  stoppered  or  well-corked,  wide-mouthed  bottle. 
Before  use  the  slide  must  be  taken  out  of  the  spirit  and 
well  rubbed  with  a  soft,  clean  linen  rag. 

Cover-glasses  are  best  sent  out  in  oil  or  covered  with 
oil,  as  even  in  the  course  of  a  voyage  lasting  only  two 
weeks  they  may  become  frosted.  The  whole  ma^  of 
cover-glasses,  say  half  an  ounce,  is  placed  in  oil  of  cloves, 
and  the  cover-glasses  are  separated  so  that  the  oil  pene- 
trates between  them.  They  are  then  taken  out  of  the 
oil,  wrapped  in  cotton-wool  and  can  be  replaced  in  then- 
boxes.  Treated  in  this  way  they  will  keep  for  months 
even  in  the  worst  climates.  They  also  keep  well  if  sent 
out  in  spirit,  but  this  is  not  recommended,  as  if  t In- 
spirit evaporates  completely  the  glasses  deteriorate  very 
quickly. 

Cover-glasses  treated  with  oil  are  not  very  easy  to 
clean,  as  the  oil  will  have  hardened  and  dried  to  a  large 
extent.  A  good  deal  of  the  oil  can  be  removed  by  plac- 
ing the  cover-glasses  in  1  per  cent,  lysol  solution  or  in 
xylol,  and  separating  them  or  stirring  them  up.  This 
saves  the  spirit  which  is  necessary  to  more  completely 
remove   the    oil.     They   must   not    be   left    more   than  an 


COVER   GLASSES 


■7 


hour  in   the  lysol,  unci   then   should  he  placed  and  kept 
in  spirit,  which  will  gradually  remove  nearly  all  the  oil. 

A  small  stock  should  be  further  prepared  so  as  to  be 
ready  for  immediate  use.  This  may  be  done  by  first  just 
bringing  them  up  to  the  boiling  point  in  a  saturated 
solution  of  carbonate  of  soda,  washing  well,  preferably 
in  running  water,  and  transferring  them  to  strong  50  per 
cent.  sulphuric  acid  ;  in  this  they  should  be  left  over 
night,  then  again  well  washed  in  water  and  finally  trans- 
ferred to  a  wide- necked,  well-stoppered  bottle  half  rilled 
with  spirit.  For  use  they  should  be  taken  out  with 
forceps  and  well  rubbed  with  a  soft  linen  rag. 


Fig.  7. 


Such  cover-glasses  should  be  free  from  both  grease 
and  grit,  and  are  then  fit  to  use  for  making  fresh  fluid 
blood-films  or  for  other  preparations.  As  alternatives  to 
the  treatment  with  carbonate  of  soda  and  sulphuric  acid, 
some  prefer  strong  nitric  acid  and  others  bichromate  of 
potash  (2  parts),  sulphuric  acid  (3  parts)  and  water  (25 
2 


iS 


si  ERILIZERS 


parts);  others,  again,  sulphuric  acid  alone.  In  any  of 
these  solutions  the  cover-glass  can  be  kept  indefinitely 
and  washed  in  water  immediately  before  use. 

Cover-glasses  should   be  of   the   best  quality,   and    for 

blood  work  the  thinnest  (No.  1)  should  be  used.  A 
smaller  stock  of  thicker  cover-classes  should  be  kept  for 
the  examination  of  faeces  and  making  "squash  "  prepara- 
tions. These  thicker  cover-glasses  do  not  deteriorate  so 
rapidly. 

For  bacteriological  work  some  form  of  steam  sterilizer 
such  as  Koch's  (fig.  7)  is  necessary  to  sterilize  vessels, 
media,  &c.    With  this  all  requisite  sterilization  for  ordinary 


Fig.  8. — Hot-air  Sterilizbr. 


work  can  be  done,  but  a  hot-air  sterilizer  is  an  advantage 
for  the  quicker  and  easier  sterilization  of  vessels,  Petri 
dishes  and  some    instruments. 

A  steam  sterilizer  (fig.  7)  is  simply  a  tall  metal  vessel 
covered  with  a  lid  with  a  vent  for  the  escape  of  steam, 
and  containing  water  at  the  bottom.  As  it  is  not  well 
to  immerse  the  objects  to  be  sterilized  in  the  water,  there 
is  a  perforated  false  bottom  above  the   level    of  the  water 


STERILIZERS 


19 


on  which  such  objects  rest.  The  whole  vessel  to  pre- 
vent loss  of  heat  is  covered  with  some  non-conducting 
material.  There  is  no  great  difficulty  in  improvising 
such  a  sterilizer,  but  those  sold  are  more  sightly  and 
convenient. 


Fig.  9. —  IIearson's  Incubator,  working  with  Petroleum  Lamp 


They  can  be  heated  by  a  paraffin  lamp,  and  the 
"Primus"  is  one  of  the  best.  At  a  pinch  a  wood  or 
other  fire  may  be  used. 

The  hot-air  sterilizer  (fig.  8)  is  a  metal  case  enclosed 
in   a   second  larger  one,   the  two   being  separated   by  an 


20  INCUBATION 

air-space.     The   double   case  can   be   dispensed  with,  but 
the  heating  is  then  less  uniform.     A  temperature  of  about 

1600  C.  is  required. 

Incubators  are  needed  where  it  is  important  that  growth 
should  take  place  at  a  uniform  temperature,  and  are 
essential  when  it  is  desired  to  describe  accurately  the 
character  of  the  growth  of  an  organism,  or  to  compare 
one  growth  with  another,  or  with  the  description  of 
another. 

Where  gas  cannot  be  obtained  a  form  of  incubator 
which    can    be   used    with    kerosine    must    be    employed 

(tig-  9)- 

At  "room  temperature"  in  the  Tropics  most  organisms 
grow  well,  and  much  useful  work  can  be  clone  without 
an  incubator. 

If  there  is  no  incubator  a  dark  cupboard  must  be  used, 
as  light  has  a  deleterious  effect  on  most  bacteria.  This 
cupboard  should  be  fixed  in  a  dry  place,  where  the  tem- 
perature is  as  uniform  as  possible. 


21 


CHAPTER  II. 
Post-mortem  Examinations. 

Post-mortem  examinations  in  the  Tropics  present  certain 
differences  from  these  examinations  in  temperate  climates. 
Post-mortem  changes  are  more  rapid,  so  that  it  is  essential 
that  the  examination  should  be  made  as  soon  as  possible 
after  death.  This  is  not  only  on  account  of  the  rapidity 
with  which  putrefactive  changes  occur,  but  also  because 
many  of  the  animal  parasites  die,  and  some,  such  as  the 
sporozoa,  disintegrate  and  cease  to  stain  well  even  before 
putrefactive  changes  set  in. 

A  large  proportion  of  tropical  diseases  are  those  affect- 
ing the  abdominal  viscera,  and  to  study  the  exact  relation- 
ship of  the  parts  it  is  often  advisable  to  remove  the 
thoracic  and  abdominal  viscera  en  masse.  The  abdomen 
should  be  freely  opened  and  room  gained  by  subcuta- 
neous division  of  the  muscles  attached  to  the  pubes 
below,  and  sternum  and  ribs  above. 

The  attachments  of  the  diaphragm  to  the  sternum  and 
costal  margins  must  next  be  divided  with  the  knife  close 
to  the  chest  wall,  and  the  parietal  peritoneum  stripped 
off  the  abdominal  wall  with  the  hand  as  far  as  possible. 
The  trachea  and  vessels  going  to  the  neck  are  then  to  be 
freely  divided  by  passing  the  hand  and  knife  in  front  of 
the  lungs  and  cutting  transversely  above  the  root  of  the 
lungs,  whilst  with  the  hand  the  thoracic  viscera  are 
grasped  firmly  at  the  root  of  the  lungs  and  steady  traction 
exercised.  When  the  division  is  complete,  the  lungs  and 
heart  will  be  easily  pulled  downwards  through  the  lower 
opening  of  the  thorax.  With  the  knife  the  posterior 
attachments  of  the   diaphragm  are  divided    from  above, 


22  POST-MORTEM    EXAMINATIONS 

and  steady  traction,  aided  by  a  few  touches  with  the 
knife,  will  strip  the  peritoneum  off  the  remainder  of  t he- 
wall  of  the  abdomen,  and  all  the  abdominal  viscera  with 
the  aorta  and  kidneys  will  he  completely  separated  except 
at  their  pelvic  attachments.  These  can  he  divided,  or 
better,  the  peritoneum  stripped  off  the  pelvis  at  each 
side,  and  the  urethra  and  rectum  divided  as  near  the 
perineum  as   possible. 

The  mass  of  organs  can  he  now  examined  from  every 
aspect  and  the  relations  of  the  different  parts  readily 
observed. 

By  this  method  the  root  of  the  mesentery,  the  posterior 
mediastinum,  and  other  parts  which  are  usually  over- 
looked can  be  displayed,  and  in  these  regions  para- 
sites are  sometimes  found. 

Certain  special  observations  are  worthy  of  attention  : — 
(i)  The  weights  and  relative  weights  of  the  organs 
vary  considerably  from  European  standards,  both  in 
health  and  disease.  With  the  lungs,  in  recording 
the  weight,  it  is  essential  to  note  also  the  time  that 
has  elapsed  between  death  and  the  examination,  as 
the  weights  of  these  organs  increase  a  few  hours 
after  death,  probably  by  aspiration  of  fluid.  If  the 
examination  is  made  two  or  three  hours  after  death 
the  lungs  will  be  barely  half  the  weight  taken  as  the 
standard  in  Europe,  whereas  if  the  examination  is 
made  later  the  weights  may  be  much  the  same.* 

(2)  Variation  in  weight  of  the  organs  with  age 
differs  in  different  races,  and  the  curves  obtained  for 
the  organs  are  in  many  cases  different  from  those 
recorded  in  Europe.  For  example,  the  brain  weight 
in  Europe  attains  its  maximum  between  45  and  50, 
whilst  in  the  negro  the  maximum  is  reached  between 
20  and  25.* 

(3)  Abnormalities  are  common,  and  some  ol  them 
occur  with   unusual   frequency  in  certain   race-,  such 

Vide  tables  in  Appendix. 


PUTREFACTIVE   CHANGES  23 

as  Meckel's  diverticulum  in  the  Chinese,  and  deeply- 
lissured  lungs  in  the  negro  races.  Diseases  also 
affect  organs  differently  according  to  race,  and  of 
this  the  age  incidence  of  splenic  enlargement  in  negro 
and  other  races  living  under  the  same  conditions  is 
a  striking  example.  While  in  childhood  all  are 
equally  affected  by  malarial  disease,  in  adult  life  the 
spleen  in  the  negro  tends  to  subside,  but  in  other 
races,  Indian,  Chinese  and  aboriginals,  it  remains 
increased  in  size,  and  is  commonly  found  post  mortem 
to  be  three  or  four  times  the  weight  of  the  normal 
organs  in  Europeans.* 

(4)  Abnormal  appearances,  such  as  congestion, 
ecchymosis,  &c,  are  more  common  in  the  Tropics, 
and  are  observed  under  different  conditions.  On 
the  one  hand,  as  the  examinations  are  made  much 
earlier  the  appearances  resemble  more  closely  those 
in  the  living  subject ;  and  on  the  other  hand,  as 
putrefactive  changes  occur  so  easily,  particularly  in 
the  vicinity  of  the  intestine,  patchy,  irregular  post- 
mortem staining  is  common,  and  is  frequently  mis- 
taken for  disease. 

(5)  Certain  special  putrefactive  changes  may  be 
a  source  of  error.  As  a  result  of  putrefaction  some 
of  the  organs,  and  particularly  the  spleen,  often 
appear  of  a  slaty  colour,  which  may  be  mistaken 
for  malarial  pigmentation.  Section  of  the  organ  will 
show  that  the  discoloration  of  early  putrefaction 
only  extends  for  a  short  distance  into  its  substance. 
The  substance  of  the  spleen  in  section  sometimes 
appears  very  dark,  but  this  colour  can  be  dis- 
tinguished from  that  of  acute  malaria  by  noticing 
that  the  dark  colour  changes  to  bright  red  after  ex- 
posure to  air.  The  only  satisfactorv  test  of  malarial 
pigmentation  of  an  organ  is  by  examination  of  a 
portion    of    the    tissue    with    the    microscope.      It    is 

*    Vide  tables  in  Appendix. 


24  EXAMINATION    FOR    ENTOZOA 

not  necessary  to  cut  sections ;  a  small  portion  of 
the  organ  can  he  forcibly  compressed  between  two 
slides  and  examined  at  once  for  pigment. 

A  diffluent  spleen  is  often  described,  but  is  not  met 
with  in  post-mortem  examinations  made  sufficiently  early 
after  death.  The  spleen,  even  in  the  most  acute  cases 
of  malaria,  though  enlarged  and  black,  is  firm,  and 
wedges  with  acute  angles  can  be  cut.  These  angles 
retain  their  sharpness  even  when  exposed  to  a  jet  of 
water.  Such  a  spleen  is  easily  forcibly  compressed,  and 
if  allowed  to  decompose  speedily  becomes  "diffluent." 

Organs,  shortly  after  death,  are  firm  and  hard  from 
rigor  mortis  of  the  tissue  elements.  When  this  passes 
off  the  organs  become  flaccid  and  softer. 

Manv  of  the  putrefactive  organisms  form  gas,  and 
consequently  emphysematous  changes  are  produced  ; 
such  emphysema  of  the  liver  and  other  organs  is  com- 
mon. In  the  intestines  small  emphysematous  patches 
form  in  the  submucosa,  and  present  a  peculiar  and  rather, 
deceptive  appearance. 

Gaseous  distension  of  the  whole  intestine  is  very 
common,  and  the  stretched  walls  appear  unusually  thin 
and  are  often  described  as  atrophied. 

EXAMINATION  FOR  ENTOZOA. —  In  the  examination  of 
intestines  in  the  Tropics  it  is  not  advisable  to  wash  out 
the  intestine  before  opening  it.  The  intestine  should 
be  opened  and  examined  for  entozoa  first  by  passing  the 
intestine  slowly  between  the  thumb  and  first  linger  so 
as  to  remove  the  intestinal  contents,  and  subsequently 
washed  to  see  the  condition  of  the  mucosa.  If  the 
intestines  are  washed  out  first,  the  entozoa  will  be 
carried  away,  and  may  escape  notice.  The  washings 
should  be  collected  and  the  deposit  examined  separately. 

Worms  and  intestinal  parasites  die  as  a  rule  within 
some  six  to  twelve  hours  after  the  death  of  the  host, 
and  some,  such  as  the  ankylostomes,  lose  their  hold  on 
the  intestinal  walls  even  earlier. 

Museum    Preparations. —  In   the  older  methods  for 


MUSEUM    PREPARATIONS  25 

the  fixation  and   hardening  of  macroscopical  specimens, 

alcohol,  and,  later,  formalin  were  employed.  These 
methods  preserved  the  form  and  relations  of  organs  well, 
but  were  practically  valueless  for  preserving  the  natural 
colours. 

Modern  methods  in  use  result  in  the  preservation  of 
the  natural  colours  of  the  specimens  and  harden  them 
at  the  same  time.  The  basis  of  the  methods  is  the  use 
of  a  first  bath  of  formalin,  which  has  the  power  of 
changing  oxyhaemoglobin  into  acid  haematin ;  of  a 
second  bath  of  spirit,  which  converts  the  acid  haematin 
into  alkali  haematin,  which  in  colour  is  very  similar  to 
oxyhaemoglobin,  so  that  the  tissues  regain  their  natural 
colours;  finally  the  specimens  are  preserved  in  a  solution 
containing  potassium  acetate,  glycerine  and  water. 

Kaiserling's  original  method,  which  gives  as  uniformly 
satisfactory  results  as  any  of  its  modifications,  is  as 
follows  : — 

(1)  The  organs  are  fixed  by  keeping  in  the  formol 
mixture  until  they  are  just  hardened — thirty-six  to 
forty-eight  hours  or  even  longer.  The  best  results 
are  obtained  by  fixing  in  the  dark. 

Formalin...          ...  ...  200  c.c. 

Water       ...          ...  ...  1,000  c.c. 

Nitrate  of  potassium  ...  15  grm. 

Acetate  of  potassium  ...  30  grm. 

As  a  substitute  10  per  cent,  formalin  may  be  used. 
Weak  formalin  must  not  be  used,  as  this  dissolves 
out  the  haemoglobin. 

A  section  of  a  solid  organ  such  as  the  spleen  or 
liver  can  be  safely  placed  in  a  vessel  containing  this 
fluid,  but  in  dealing  witii  softer  tissues  it  is  advisable 
to  wrap  the  specimen  loosely  in  cotton-wool  before 
immersion,  and  to  give  it  plenty  of  room.  In  dealing 
with  an  entire  organ  it  is  best  to  inject  it  with  the 
fluid,  as  the  organ  then  retains  its  shape  better,  and 
the  haemoglobin  in  its    interior    is   more  completclv 


26  MICROSCOPIC    PREPARATIONS 

converted,  and    docs    nol    afterwards    leak   oul  and 

colour  the  mounting  medium. 

(2)  After  fixation  the  specimen  is  placed  ill  the 
second  bath,  which  consists  of  spirit.  It  was  origin- 
ally recommended  to  employ  increasing  strengths 
of  spirit,  but  equally  good  results  are  obtained  by 
placing  the  specimen  at  once  in  90  per  cent,  spirit. 
The  length  of  time  necessary  for  this  bath  must  be 
determined  by  the  appearance  of  the  specimen  ;  it 
Should  be  removed  when  the  original  colour  fully 
returns,  which  is  usually  in  twenty-four  to  thirty-six 
hours. 

(3)  Mount  in  jars  containing  :— 

Glycerine         ...  ...        400  c.c. 

Acetate  of  potassium...        200  grra. 
Water  ...  ...  ...     2,000  c.c. 

A    few    crystals    of    thymol  or  a    trace    of    formalin 
may  be  added  to  prevent  the  growth  of  moulds. 

Preparatiox  of  Tissues  for  Microscopic 
Exam  ix  ati  ox. 

Parts  of  an  organ  or  tissues  kept  for  microscopical 
examination  may  be  examined  fresh  or  preserved  and 
hardened. 

From  the  examination  of  the  fresh  specimen  much 
information  may  be  gained.  Smears  of  the  fluids  that 
exude  from  the  cut  surface  may  be  made,  small  portions 
of  the  tissue  may  be  squashed  between  a  slide  and  cover- 
glass,  or  the  material  may  be  frozen  and  sections  made. 
In  the  last  case  treatment  in  a  strong  solution  of  gum 
arabic  is  desirable.  The  details  of  the  methods  useful 
for  the  determination  of  parasites  is  considered  with  the 
description  of  these  parasites.  It  is  from  the  fresh 
specimens  that  cultures  must  be  made.  For  this  pur- 
pose the  spleen  and  lymphatic  glands  are  the  most 
suitable  places.  Blood  should  be  withdrawn  by  a  sterile 
syringe   from  the   unopened    heart  after   First    searing  the 


FIXATION   AND    HARDENING  27 

surface    with    a    hot    iron    for    culture-making  and    for 

inoculation   of  animals. 

Fixation  and  Hardening. — Although  formalin  is  com- 
monly recommended  as  a  fixing  fluid  when  ordinary 
tissue  changes  are  to  be  demonstrated,  it  will  be  found 
in  tropical  practice  that  alcohol  is  the  most  generally 
useful  reagent,  as  protozoal  and  bacterial  organisms  stain 
better  after  fixation  by  this  method.  It  has  the  disadvan- 
tage of  causing  great  shrinking  of  the  tissues  from  rapid 
dehydration. 

To  fix,  the  tissue  should  be  first  placed  in  80  per  cent, 
alcohol  and  in  two  to  four  hours  transferred  to  95  per 
cent,  alcohol.  The  tissue  must  be  in  small  cubes  not 
more  than  half  an  inch  in  their  greatest  length,  and 
placed  in  at  least  ten  times  their  volume  of  alcohol  in  a 
closed  glass  vessel. 

If  a  piece  of  tissue  is  simply  put  into  a  bottle  and  spirit 
poured  on  it,  the  blood  coagulates  at  the  edges  where  it  is 
in  contact  with  the  glass,  and  the  fluid  does  not  penetrate 
between  the  glass  and  tissue.  This  is  avoided  by  placing 
some  cotton-wool  or  small  pieces  of  crumpled  paper  at 
the  bottom  of  the  bottle  before  introducing  the  piece  of 
tissue. 

In  certain  cases,  especially  where  it  is  desirable  that 
the  blood  should  be  retained  in  the  vessels,  a  larger 
piece  of  tissue  can  be  taken,  and  after  partial  fixation 
subdivided  into  pieces  of  the  right  size.  This  is  par- 
ticularly to  be  recommended  when  the  object  is  the 
examination  of  the  tissue  for  malaria  parasites  or  filaria 
in  si  hi.  At  the  end  of  six  hours  the  alcohol  should  be 
changed,  and  again  changed  in  twelve  hours.  By  this 
time  in  a  warm  climate  the  specimen  will  be  sufficiently 
fixed,  and  longer  immersion  in  strong  alcohol  will 
render  the  specimen  too  brittle.  In  colder  weather, 
where  the  average  temperature  is  under  70°  F.,  it  can  be 
left  for  some  hours  longer  in  the  alcohol. 

The  specimens  when  fixed  can  be  kept  in  methylated 
spirit  till  required.     If  greater   accuracy  is  required   the 


28  FIXATION    AND    HARDENING 

specimens  can  be  kept  in  no  per  cent,  absolute  alcohol. 
This  will  keep  the  specimens,  and  stronger  alcohol  at 
tropical  temperatures  soon  overhardens  them. 

FORMOL  ALCOHOL. — For  the  more  rapid  fixation  of 
tissues  where  examination  for  malaria  parasites  is  not 
required,  alcohol  and  formalin  give  excellent  results.  This 
solution  is  made  by  the  addition  of  formalin  in  the  pro- 
portion of  2  to  10  per  cent,  to  the  absolute  alcohol.  It 
penetrates  rapidly  and  causes  less  shrinking  than  alcohol 
alone,  but  the  tissues  should  not  be  left  in  this  solution 
for  more  than  twelve  hours  or  they  will  be  overhardened. 
They  are  then  fit  for  further  processes  or  can  be  kept  in 
spirit. 

MtJLLER'S  Fluid. — Pot.  bichromate  2-5  parts,  sodium 
sulphate  1  part,  and  water  100  parts  is  very  extensively 
used  and  gives  good  results,  but  is  slow  in  its  action. 
The  pieces  of  tissues  are  placed  in  an  abundance  of 
the  fluid,  which  should  be  changed  in  a  few  hours,  and 
again  daily  for  a  week,  after  that  once  a  week  will  be 
sufficient.  Some  tissues  will  be  sufficiently  fixed  in  two 
or  three  weeks,  but  others,  as  the  parts  of  the  central 
nervous  system,  may,  even  in  a  warm  tropical  climate, 
require  many  weeks.  When  fixation  is  complete  the 
specimens  should  be  washed  for  twenty-four  hours  in 
abundance  of  water,  which  is  frequently  changed,  pre- 
ferably in  running  water,  and  then  kept  in  methylated 
spirit. 

Animal  parasites  do  not  stain  well  in  tissues  which 
have  been  fixed  in  bichromate  solutions. 

Orth's  Fluid. — Miiller-formol  is  made  by  adding  10 
.per  cent,  of  formalin  to  Miiller's  fluid.  This  must  be 
added  immediately  before  use.  It  is  a  rapid  fixative, 
and  at  blood  heat  only  three  or  four  hours  are  required 
for  thin  pieces  of  tissue.  Two  days  are  usually  sufficient 
at  room  temperature. 

When  hardened  the  tissues  may  be  cut  directly  on 
the  freezing  microtome,  or  after  thorough  washing 
passed  through  increasing  strengths  of  alcohol  and  then 
imbedded. 


IMBEDDING  2Q 

Zenker's  Fluid. — For  the  examination  of  skin,  which 
is  readily  overhardened,  Zenker's  fluid  gives  good  results. 
This  is  composed  of  5  parts  of  corrosive  sublimate,  2*5 
parts  of  potassium  bichromate,  1  part  of  sodium  sulphate, 
5  parts  glacial  acetic  acid,  and  100  parts  of  water.  The 
slices  of  tissue  to  be  examined  must  be  very  thin,  not 
more  than  a  tenth  of  an  inch  in  thickness.  The  time 
required  for  fixation  is  twelve  to  twenty-four  hours, 
according  to  the  thickness  of  the  specimen  and  the 
temperature. 

After  the  tissues  are  fixed  they  must  be  thoroughly 
washed  in  water,  which  is  frequently  changed,  for  at  least 
twelve  hours,  and  should  then  be  placed  in  spirit  to 
which  a  little  tincture  of  iodine,  or  a  few  drops  of  Gram's 
iodine  solution,  has  been  added,  to  remove  any  mercury 
deposited  in  the  tissues.  If  the  colour  of  iodine  dis- 
appears from  the  fluid  more  iodine  is  to  be  added  until 
the  colour  no  longer  disappears.  The  specimen  can  then 
be  kept  in  spirit  till  required  for  use.  Or  the  specimens 
can  be  kept  in  spirit  and  the  cleaning  with  iodine  done 
after  the  sections  are  cut. 

Osmic    Acid    Mixtures. — Two   other   useful    fixative 
agents  are  Flemming's  solution  : — 

Chromic  acid  1  per  cent.,  aqueous  solution  15  c.c. 
Osmic  acid  2  per  cent.,  aqueous  solution...    4  c.c. 
Glacial  acetic  acid     ...  ...  ...  ...     1  c.c. 

and  Hermann's  solution,  in  which  1  per  cent,  solution  of 
platinum  chloride  is  substituted  for  the  1  per  cent,  solu- 
tion of  chromic  acid  in  Flemming's  solution. 

These  solutions  must  be  freshly  made  up  before  use, 
and  as  the  penetrating  power  of  the  fixative  is  low,  the 
specimens  must  be  very  thin,  not  more  than  one-twelfth 
of  an  inch  in  thickness. 

Fixation  takes  from  one  to  two  days,  and  the  speci- 
mens require  to  be  thoroughly  washed,  preferably  in 
running  water,  for  one  day,  and  then  placed  in  80  per 
cent,  alcohol. 

Boiling  Method. — For  the  rapid  fixation  of   tissues 


30  IMBEDDING 

they  m. iv  be  placed  in  water  that  lias  been  heated  just 
to  the  boiling  point.  II  small  pieces  ol  tissue  are  used 
a  lew  minutes  will  suffice  to  coagulate  the  albumin-. 
This  method  is  also  useful  for  the  examination  of  renal 
casts  and  for  the  contents  of  cysts. 

Imbedding.  —  The  tissues  having  now  been  fixed, 
hardened,  and  completely  dehydrated,  are  assumed  to 
be  in  absolute  alcohol.  Sections  of  hardened  tissues 
can  be  cut  with  a  razor  by  hand,  or  with  a  microtome 
knife  after  fastening  the  specimen  in  the  microtome 
clamp.  Fair  sections  ot  firm  tissues  can  sometimes  be 
obtained  in  this  way  or  by  means  of  the  freezing  micro- 
tome. With  these  methods,  however,  portions  of  the 
tissue  are  likely  to  fall  out  of  the  sections,  and  it  is  desir- 
able to  have  the  tissues  imbedded  in  some  material  with 
which  they  become  permeated  and  which  preserves  the 
component  parts  in  their  relative  positions  and  surrounds 
them  with  a  protective  coating.  They  can  then  be  cut 
into  thin  sections  on  a  microtome. 

The  two  substances  in  common  use  for  imbedding 
purposes  are  paraffin  ai\d  celloidin.  Paraffin  imbedding 
is  the  most  useful  if  very  thin  sections  are  desired.  For 
certain  purposes  celloidin  is  indispensable,  as  when  it 
is  desired  to  keep  any  loose  bodies  in  situ  in  a  tissue, 
there  being  no  necessity  to  remove  the  celloidin  before 
mounting  in  Canada  balsam. 

Hard  tissues,  and  tissues  which  easily  become  brittle, 
such  as  muscle  and  skin,  are  cut  with  considerable  diffi- 
culty by  the  paraffin  method. 

(i)  Paraffin  Lyibfddixg. — The  general  principle  is 
to  pass  the  specimen  through  alcohol  till  it  is  thoroughly 
dehydrated,  then  to  place  it  in  a  fluid  in  which  paraffin 
is  soluble,  which  will  dissolve  out  the  alcohol,  and  then 
to  replace  this  fluid  by  first  a  weak  solution  of  paraffin, 
then  a  strong  solution  of  paraffin,  and  finally  melted 
paraffin  wax.  Excess  of  paraffin  is  poured  round  the 
tissue,  and  it  is  allowed  to  cool  ;  when  the  paraffin  solidities 
not  only  is  the  piece  ol  tissue  enclosed  in  a  solid  block  of 


IMBEDDING  31 

paraffin   wax   but  the  tissues   will   be   permeated  with  the 


wax. 


There   are    many    modifications,    some    of    which    are 
rendered  necessary  for  special  tissues. 


Fig.  10. 


Fig.   11. 


Fig.    12. 


For  general  work  with  specimens    taken    from  strong 
spirit : — 

(1)  Place  the  specimen  in  absolute  alcohol  for 
twenty-four  hours.  If  the  specimen  has  been  re- 
moved from  weaker  spirit  or  from  water,  before 
placing  in  the  absolute  alcohol  it  should  be  placed 
in  methylated  spirit  for  forty-eight  hours. 

(2)  Remove  from  spirit,   dram   off  excess  of  spirit 


32 


RAPID    PARAFFIN    METHOD 


for  ;i  few  minutes  and  place  in  aniline  oil.     One  day. 

(3)    Place  in  xylol.      One  daw 

(_})  Place  in  paraffin  and  xylol,  equal  parts.  One 
day. 

(5)  Place  in  melted  paraffin  wax  tor  one  day.  The 
paraffin  wax  can  be  kept  melted  in  a  drying  oven 
(tig.  10)  at  the  required  temperature,  or  a  paraffin 
embedding  bath  can  be  used  for  this  purpose  die;.  11). 
As  a  considerable  amount  of  spirit  is  required  for 
the  spirit  lamp  to  maintain  the  required  temperature, 
it  is  well  to  imbed  as  many  specimens  as  possible  at 
the  same  time.     The  imbedded  specimens  keep  well. 

(6)  Imbed  and  cool  quickly. 

The  imbedding  may  be  done  by  tilling  small  paper 
boxes  with  melted  paraffin  and  placing  the  pieces  of 
tissue  in  this  melted  paraffin.  The  box  is  then  placed 
in  a  dish  of  cold  water  on  which  it  floats  and  is  rapidly 
cooled,  so  that  the  paraffin  sets  without  crystallizing. 
Or  L-shaped  pieces  of  metal  are  placed  in  contact  on 
a  smooth  slab,  as  in  the  diagram  (tig.  12),  and  the  space 
between  rilled  with  the  melted  paraffin  and  the  specimens 
placed  in  as  before. 

Modifications. — The  paraffin  used  in  England  melts  at 
too  Iowa  temperature  for  satisfactory  work  in  the  Tropics. 
It  is  well  therefore  to  keep  two  varieties  of  paraffin,  one 
melting  at  480  C.  and  the  other  at  6o°  C,  and  to  use 
a  mixture  of  them.  Such  a  mixture  with  a  melting 
point  about  54°  C.  is  usually  sufficient,  but  in  the  warmest 
weather  either  a  larger  admixture  of  the  paraffin  at  the 
higher  melting  point  will  be  required,  or  the  pure  paraffin 
melting  at  6o°  C. 

Rapid  Paraffin  Imbedding  Method. — For  the  rapid 
examination  of  small  objects  the  process  of  imbedding 
may  be  shortened  by  the  use  ot  acetone,  which  hardens 
and  dehydrates  the  tissue  and  at  the  same  time  prepares  it 
for  immersion  in  paraffin. 

(1)   Fix  small  pieces  ot   tissue  in   10  per  cent,  for- 
malin for  one-hall  to  lour  hours. 


CELLOIDIN    IMBEDDING  33 

(2)  Place  in  pure  acetone  for  one-half  to  one  and  a 
half  hours. 

(3)  Transfer  directly  to  fluid  paraffin  (520  to 
560  C),  and  place  in  the  oven  for  one-half  to  one 
and  a  half  hours.  The  acetone  evaporates  and  the 
paraffin  permeates  the  tissues. 

(4)  Prepare  the  paraffin  block.  Cut  and  stain  as 
usual. 

The  acetone  may  be  used  again  by  placing  fired  copper 
sulphate  at  the  bottom  of  the  vessel. 

(2)  Celloidin  Imbedding.  —  Commercial  celloidin 
(Schering)  is  a  purified  gun-cotton.  It  is  sold  in  granules, 
in  shavings,  or  in  flat  slabs.  The  latter  require  to  be  cut 
into  small  squares  before  using. 

To  imbed  in  celloidin,  the  general  principle  is  the  same 
as  that  for  paraffin,  but  the  agents  employed  and  the 
methods  differ. 

(1)  The  specimen  is  kept  in  absolute  alcohol,  after 
being  in  weaker  spirit,  for  twenty-four  hours. 

(2)  It  is  then  soaked  in  a  mixture  of  equal  parts 
of  ether  and  absolute  alcohol  for  twenty-four  hours. 

(3)  Place  in  a  weak  solution  of  celloidin  (3  per 
cent.)  in  alcohol  and  ether  for  twenty-four  hours  or 
more  ;  two  days  is  usually  ample. 

(4)  It  is  then  to  be  transferred  to  a  thicker  celloidin 
solution,  6  per  cent,  celloidin  dissolved  in  alcohol 
and  ether,  and  kept  in  this  for  at  least  one  clay,  and 
better  for  several  days. 

(5)  The  specimen  is  then  placed  on  a  small  block 
of  wood,  on  which  a  few  drops  of  the  thick  celloidin 
have  been  placed.  Leave  exposed  to  the  air  for  a 
tew  minutes,  and  pour  a  little  thick  celloidin  solution 
over  the  specimen.  Expose  to  air  for  a  few  minutes 
until  a  whitish  film  appears  on  the  surface,  and  place 
in  60  per  cent,  alcohol,  which  will  harden  the  cel- 
loidin. In  cutting  celloidin  specimens,  the  knife 
must  be  oblique,  and  must  be  kept  constantly 
moistened  with  spirit. 


34 


skctm  )\-ri'i  rixc 


Section-Cutting.  —  For  this  purpose  ;i  microtome  is 
required,  and  the  greater  number  of  those  available  may 
be    used    for  either  celloidin   or   paraffin.      For   cutting 

frozen  sections  a  special  instrument  is  necessary. 

Cathcart's  microtome  (tig.  13)  is  the  simplest  efficient 
freezing  microtome,  but  in  the  Tropics  its  use  is  neces- 
sarily limited,  as  freezing  with  ether  is  not  practicable 
in  most  tropical  countries. 


Fig.  13.— Microtome,  Cathcart's,  with  Spray  Bellows. 


Imbedded  sections  can  be  cut  with  this  microtome, 
but  must  not  be  frozen.  The  carrier  is  heated  and  the 
paraffin  block  pressed  against  it.  The  paraffin  will  be 
melted,  and  will  then  adhere  to  the  zinc  plate  ;  but  better 
sections  can  be  obtained  with  other  microtomes. 


FRFEZIN'G   MICROTOME 


35 


For  freezing  in  the  Tropics  a  mixture  such  as  ice  and 
salt  is  the  best. 

Swift's  microtome  consists  of  a  circular  wooden  box 
(figs.  14,  15)  (a)  from  the  centre  of  which  rises  a  metal 
tube  surmounted  by  a  horizontal  zinc  plate  raised  above 
the  level  of  the  top  of  the  box.  The  box  is  covered 
with  a  glass  plate  perforated  in  the  centre  with  a  hole 
large  enough   to  allow  the  tube   and  zinc    plate  to  pass 


Fig.    14. 


Fig.    15. 


through.  When  arranged  for  use,  the  box  is  filled  with 
a  mixture  of  well-crushed  ice  and  salt.  The  lid  is  placed 
on  and  the  zinc  plate  projects  above  its  level.  The 
substance  to  be  frozen  is  placed  on  the  zinc  plate  and 
well -covered  with  a  strong  solution  of  gum.  If  the  air 
temperature  is  not  too  high,  the  specimen  with  the 
surrounding  gum  freezes. 

In  many  tropical  countries  this  does  not  suffice  unless 
the  specimen  is  also  surrounded  by  a  cold   atmosphere. 


36  FREEZING   MICROTO.Mi: 

This  is  done  by  placing  ;i  second  metal  box  (fig.  15,  H) 

on  the  top  of  the  glass  plate.  This  metal  box  has  a 
central  tube  rising  bom  the  bottom  and  open  below, 
and  this  tube  must  be  wide  enough  to  enclose  the  zinc- 
plate  and  specimen.  If  the  metal  box  is  also  filled  with 
the  freezing  mixture,  the  air  in  the  central  tube  will  be 
cold,  and  the  specimen  surrounded  by  this  cold  air  freezes 
readily. 

When  the  specimen  is  frozen,  the  upper  metal  box  can 
be  removed,  and  sections  cut. 

In  this  instrument  the  specimen  remains  fixed,  and 
the  thickness  of  the  section  is  regulated  by  alterations 
in  the  level  of  the  razor.  This  is  arranged  by  having 
the  razor  blade  fixed  on  a  tripod  ;  the  length  of  the  legs 
of  this  tripod  can  be  regulated  by  turning  the  milled 
heads  of  the  screws.  The  feet  of  the  tripod  are  tipped 
with  bone  so  as  to  slide  evenly  over  the  glass.  For 
use  the  blade  of  the  razor  must  be  wetted  with  water, 
and  the  tripod  carrying  it  is  so  arranged  that  one  leg  is 
anterior.  The  two  posterior  screws  are  turned  till  the 
edge  of  the  razor  is  horizontal  or  parallel  with  the  sur- 
face of  the  glass.  Any  alteration  in  the  screw  of  the 
anterior  leg  will  then  raise  or  lower  the  edge  of  the  blade. 

The  sections  are  cut  by  gliding  the  tripod  over  the 
plate  till  the  edge  of  the  razor  touches  the  specimen,  and 
cuts  through  it.  A  slightly  oblique  motion  is  the  best, 
and  the  tripod  must  be  pressed  firmly  on  the  glass  plate 
and  the  movements  must  be  rapid,  more  like  a  thrust. 
The  knack  of  making  the  correct  movement  is  soon 
acquired. 

When  a  section  is  cut,  the  tripod  is  drawn  back,  slightly 
tilting  it  to  avoid  touching  the  specimen,  the  anterior 
screw  turned  to  an  amount  regulated  by  the  thickness 
of  the  desired  section,  and  again  thrust  forward.  This 
process  should  be  repeated  until  there  are  several  sections 
on  the  upper  surface  of  the  blade  of  the  razor,  and  these 
can  be  removed  with  a  camel's  hair  brush  to  a  vessel  con- 
taining water  which  has  been  recently  boiled,  so  as  to  be 


PARAFFIN    SECTIONS 


37 


free  from  air.  The  sections  will  float,  and  can  be  floated 
on  to  a  slide,  and  either  examined  directly  or  stained. 

Sections  of  fresh,  unfixed  tissues  can  be  cut  and 
examined  unstained,  or,  to  show  structure  better,  they 
can  be  stained.  They  should  be  soaked  in  gum  before 
freezing. 

Well-fixed  specimens  can  also  be  frozen  and  cut.  It 
is  necessary  before  freezing  to  thoroughly  remove  the 
last  traces  of  alcohol  by  washing  in  water,  and  to  soak 
in  an  aqueous  solution  of  gum  arabic  for  some  hours. 
Where  ice  cannot  be  obtained  further  hardening  and 
imbedding  is  necessary. 


Fig.  16. — Cambridge  Rocking  Microtome,  new  Pattern  for 
cutting  flat  sections,  with  large  articulating  apparatus  and 
one  razor. 


Paraffin  Sections. — Of  the  simpler  and  cheaper 
forms  of  microtome  suitable  for  cutting  paraffin  sections, 
the  Cambridge  Rocker  is  the  most  convenient. 

Full  directions  for  the  use  of  this  instrument  are  sent 

with  the  microtome,  but  the  chief  points  to  observe  are  : — 

(i)  That  the  razor  must  be  rigidly  clamped. 

(2)  That  the  paraffin  block  must  be  firmly  fixed  on 

the    metal    carrier.     This    is    done   by   heating   the 


38  CELLOIDIN   SECTIONS 

carrier  and  applying  the  paraffin  block  firmly  to  it, 
and  keeping  it  in  position  till  the  carrier  is  cold. 

(3)  That  the  thickness  of  section  must  be  graduated 

in  accordance  with  the  nature  of  the  tissue,  its  brittle- 

ness,  and  object  of  the  section.     If  the  specimen  is 

too  hard  or  brittle  it  is  useless  to  expect  thin  sections. 

Sections  showing  large  parasites,  such  as  filaria,  in  tissues 

like  the  lung,  should  not  be  too  thin.     For  the  details  of 

nerve  structure,  and  for  sections  showing  bacilli,  or  the 

parasites  of  malaria,  the  thinnest  possible  sections  are  the 

best. 

Celloidix  Sections. — For  cutting  celloidin  sections 
a  slide  microtome,  such  as  that  of  Jung,  is  best,  but  quite 
good  sections  may  be  obtained  with  the  Cambridge 
Rocker.  A  form  of  this  instrument  should  be  selected 
in  which  the  razor  can  be  placed  obliquely. 

These  sections  are  generally  cut  in  alcohol.  The  sur- 
faces of  the  celloidin  block  and  of  the  knife  should  be 
moistened  with  alcohol  by  means  of  a  camel's  hair  brush. 
Place  the  knife  so  that  it  forms  a  slight  angle  with  the 
block.  Transfer  the  cut  section  to  70  per  cent,  alcohol 
with  a  brush  or  with  the  finger. 

From  paraffin  blocks  sections  5  to  7  //,  in  thickness  may 
be  obtained  ;  from  celloidin  blocks  sections  10  to  15  /x  are 
generally  cut. 

Fixation  of  Paraffin  Sections  on  Slide. — For  ordinary 
work  the  sections,  when  cut,  are  placed  on  the  surface 
of  some  warm  water  at  about  440  C,  or  rather  more  if 
paraffin  of  a  higher  melting  point  is  used,  in  order  to 
straighten  them  out,  and  can  then  be  floated  on  to  a 
slide.  The  water  is  allowed  to  drain  off,  and  the  slide  is 
then  placed  in  the  hot  incubator  for  twelve  to  fifteen 
hours.     The  section  will  then  be  fixed  to  the  slide. 

Occasionally  it  will  be  found  that  the  sections  after 
removal  of  the  paraffin  fall  off.  In  such  a  case  the  other 
sections  may  be  very  gently  warmed  over  a  flame  till  the 
paraffin  begins  to  appear  more  translucent  and  then 
allowed  to  cool. 


FIXATION    AND   TREATMENT   OF   SECTIONS  39 

Another  method  is  to  employ  a  mixture  of  albumin 
and  glycerine  to  fix  the  section.  This  mixture  is  pre- 
pared by  beating  up  the  white  of  an  egg,  filtering,  and 
adding  to  it  one  third  the  quantity  of  glycerine.  A  crystal 
of  carbolic  acid  or  thymol  may  be  added  to  prevent  the 
growth  of  moulds.  A  thin  film  of  the  mixture  is  placed 
on  a  slide,  and  on  to  this  the  paraffin  section  previously 
straightened  out  on  the  surface  of  warm  water  is  floated. 
As  before,  the  slides  are  placed  in  the  hot  incubator  after 
draining  off  the  superfluous  water. 

Paraffin  sections  can  be  rapidly  fixed  by  floating  them 
out  on  a  drop  of  water  placed  on  the  slide.  The  slide  is 
warmed  till  the  section  flattens  out,  and  the  water  is  then 
drained  off  and  the  section  firmly  pressed  to  the  slide 
with  a  piece  of  clean  blotting  paper.  This  should  leave 
the  dried  section  closely  applied  to  the  dried  slide.  The 
slide  is  now  again  heated  until  the  paraffin  begins  to 
appear  translucent,  and  again  firmly  pressed  to  the  slide. 
This  method  should  not  be  used  for  delicate  tissues  such 
as  the  brain,  as  the  firm  pressure  required  is  harmful. 

Treatment  of  the  Sections. — To  remove  paraffin  from 
the  sections  so  that  aqueous  and  other  stains  can  be 
used,  the  slide  carrying  the  section  should  be  placed 
in  xylol  and  agitated  in  it  for  two  or  three  minutes- 
This  dissolves  the  paraffin.  To  remove  the  xylol  place 
in  strong  spirit  or  absolute  alcohol  and  again  agitate,  so 
that  fresh  surfaces  of  spirit  are  brought  in  contact  with 
the  section.  As  a  precaution  it  is  well  to  rinse  in  fresh 
spirit.  The  slide  can  then  be  placed  in  water  to  remove 
the  spirit,  and  stained  as  is  considered  advisable.  If  the 
tissue  has  been  hardened  in  corrosive  sublimate  it  should 
be  rinsed  in  iodine  solution  and  again  washed  before 
staining. 

After  staining,  dehydrate  in  alcohol,  clear  in  oil  of 
cloves,  wash  with  xylol  if  aniline  stains  are  used,  and 
mount  in  xylol  Canada  balsam.  If  the  alcohol  used  is 
strong  enough  the  oil  of  cloves  need  not  be  used. 


40 


CHAPTER   III. 
Blood. 

Examination  of  the  blood  is  of  such  importance  in 
tropical  work  that  in  all  cases  of  difficulty  and  doubt 
recourse  to  this  method  of  diagnosis  is  essential.  A 
thorough  knowledge  of  the  constituents  of  normal  blood 
is  a  necessary  preliminary.  The  abnormal  forms  of  cells 
met  with  in  various  diseases  must  be  readily  recognized. 
Last,  but  not  least,  the  various  methods  used  for  the 
finding  and  recognition  of  parasites  must  be  known. 
Many  methods  of  examining  blood  have  been  employed 
and  most  of  them  are  good.  Fallacies  and  mistakes 
have  occurred  with  all,  and  the  sources  and  causes  of 
these  errors  and  the  recognition  of  them  have  to  be 
studied.  These  are  dealt  with  under  each  method 
described. 

Blood  is  composed  of  a  nearly  colourless  fluid,  the 
plasma,  in  which  are  floating  cellular  elements,  the  red 
and  white  blood  corpuscles  and  blood-plates.  The  more 
solid  elements,  the  blood  corpuscles,  will  be  considered 
first.  They  vary  in  number,  in  their  relative  proportions 
and  in  their  characters. 

Other  cells,  not  normally  present  in  the  blood,  are 
found  under  certain  conditions  in  that  fluid.  Most  of 
these  cells  are  normally  present  in  the  tissues  of  the 
healthy  body,  though  not  normally  in  the  blood. 

Parasites  occur  in  the  red  corpuscles  and  in  the  plasma, 
and  are  sometimes  found,  in  a  more  or  less  disorganized 
condition,  in  white  corpuscles  or  phagocytes,  which  have 
devoured  them.  None  have  been  observed  in  the  blood- 
plates. 


BLOOD    FILMS  41 

The  two  main  methods  employed  for  the  examination 
of  blood  are  : — 

(1)  In  the  fresh  and  fluid  condition. 

(2)  As  films  which  are  allowed  to  dry,  and  fixed 
and  stained  in  various  ways. 

These  two  methods  are  of  general  application.  For 
special  purposes,  so  as  to  reveal  abnormal  bodies  scantily 
present  in  the  blood,  thick  films  can  be  made  and  the 
haemoglobin  removed.  In  this  way  a  quantity  of  blood 
that  would  not  be  sufficiently  transparent  if  treated  by 
the  ordinary  methods  can  be  rapidly  examined. 

Examination  of  Fresh  Blood. — This  method  is  the 
only  one  by  which  vital  changes  can  be  observed.  Of 
the  normal  blood  elements,  the  amoeboid  and  phagocytic 
properties  of  the  leucocytes  can  thus  be  observed. 
Living  organisms  abnormally  present,  such  as  filarial, 
trypanosomes,  and  the  parasites  of  malaria,  can  be 
watched,  and  such  developmental  and  degenerative 
changes  as  occur  in  shed  blood  observed. 

No  description  or  observation  of  new  parasites  is  com- 
plete without  an  examination  of  these  parasites  in  the 
living  conditions.  It  is  noteworthy  that  most  of  the 
important  mistakes  made  even  by  experienced  observers 
in  the  description  of  bodies  met  with  in  blood  have  been 
due  to  neglect  of  the  examination  of  fresh  fluid  blood. 

The  essential  point  in  the  preparation  of  fresh  fluid 
blood  films  is  that  a  great  part  of  the  film  should  be  so 
thin  that  the  blood  corpuscles  are  lying  flat  and  separate 
from  each  other. 

(1)  The  simplest  method  of  making  such  a  film  is  to 
take  a  small  drop  of  blood  on  the  centre  of  a  cover-glass 
and  drop  it  on  the  slide  (fig.  17).  In  a  well-made  film  by 
this  method  three  zones  are  apparent.  The  edge  of  the 
film  is  thick  and  irregular.  Here  the  corpuscles  are  in 
rolls  or  masses,  and  it  is  too  thick  for  the  examination  of 
the  individual  red  corpuscles  (fig.  17,  a). 

Internally  to  this  is  an  area  with  a  slightly  opaque  or 
ground-glass  appearance.      Here  the  red  corpuscles  will 


42 


FRESH    FILMS 


be  found  lying  flat  and  not  to  any  great  extent  over- 
lapping each  other.  This  is  the  part  of  the  film  best 
suited  for  examination  of  the  red  corpuscles  and  the 
parasites  contained  in  them  (fig.  17,  e). 

The  centre  of  the  film  is  clear  and  transparent,  and 
here  lew  corpuscles  are  found,  as  this  part  is  composed 
almost  entirely  of  the  plasma  (fig.  17,  b). 


0  V 

c 

0 

b 

Fig.    17. 

o 


a 


To  get  good    films   by  this  method    there  are  certain 
points  to  be  observed  :  — 

(1)  The  slide  and  cover-glass  must  be  free  from 
grease,  otherwise  the  blood  will  not  spread  out  (vide 
cleaning  slides  and  cover-glasses). 

(2)  They  must  be  freed  from  grit ;  this  is  best 
done  by  rubbing  well,  immediately  before  use,  with 
a  soft  linen  rag. 

(3)  The  drop  of  blood  must  be  so  small  that,  when 
spread  out,  it  does  not  extend  to  the  edges  of  the 
cover-glass.  If  the  blood  is  too  abundant  it  floats 
up  the  cover-glass,  and  sufficient  space  is  left  between 
the  slide  and  cover-glass  to  allow  of  the  formation 
of  rouleaux. 

(2)  Another    method    is    that    of    Braddon.     Here   the 


FRESH    FILMS 


43 


square  or  oblong  cover-glass,  freed  from  grease  and  grit, 
is  placed  on  a  slide  similarly  cleaned.  The  cover-glass 
is  so  placed  that  its  edge  corresponds  with  one  edge  of 
the  slide.  Pressure  is  exercised  on  the  centre  of  the 
cover-glass,    or    it    can    be    fixed   with    Cornet    forceps. 


Fig.    18. 


Vaseline  is  then  applied  to  the  slide  at  the  edges  of  the 
cover-glass,  leaving  the  side  applied  to  the  edge  of  the 
slide  and  a  small  space  at  the  edge  opposite  to  this  free 
(fig.  18).     The  Cornet  forceps  are  then  removed. 

These  slides  can  be  prepared  in  the  house  or  laboratory 
and  are  then  ready  for  use.     If  the  edge  of  the  slide  be 


44  EXAMINATION   OF    FILMS 

applied  to  a  drop  of  blood,  the  blood  will  run  up  by 
capillary  attraction   and  spread   itself   but   in   the  space 

between  the  cover-glass  and  slide  in  a  film  thin  enough 
for  examination. 

(3)  A  third  method  is  to  make  the  film  between  two 
cover-glasses,  the  lower  one  being  much  the  longer.  The 
blood  spreads  more  readily  between  two  cover-glasM> 
than  between  a  slide  and  cover-glass,  and  the  free  edge 
of  the  lower  glass  can  be  clamped  on  to  the  slide 
(Horder's  method).  Beautiful  films  are  obtained,  and 
cover-glasses  can  be  carried  in  larger  numbers  than  slides 
on  account  of  the  smaller  weight,  but  the  greater  fragility 
of  cover-glasses  is  a  serious  objection  to  the  general 
adoption  of  this  method. 

All  of  the  above  methods  give  good  results.  The  first 
has  decided  advantages  in  that  the  blood  elements  are  all 
present  and,  to  some  extent,  distributed  evenly  through- 
out the  best  part  of  the  film.  The  second  is  very  con- 
venient for  class  work,  as  there  is  no  delay  at  the  bedside, 
and  a  large  number  of  preparations  can  be  made  quickly. 
It  is  useful  with  nervous  patients,  as  no  preparation  is 
necessary  at  the  bedside.  The  more  adhesive  elements 
of  the  blood,  the  blood-plates  and  leucocytes,  are  crowded 
together  near  the  edge  where  the  blood  has  entered.  In 
the  thinner  part  of  the  field,  which  is  farther  from  the 
edge,  these  elements  have  been  "  filtered  out  "  and  few 
solid  elements  but  the  red  corpuscles  are  left. 

In  the  freshly  drawn  blood  the  elements  normally 
present  are  : — 

(1)  Red  corpuscles  or  erythrocytes. 

(2)  White  corpuscles  or  leucocytes. 

(3)  Blood-plates    or   platelets,    Hayem's    haemato- 
blasts. 

(4)  Plasma. 

In  the  red  corpuscles  the  points  to  note  are  the  colour, 
the  size  and  the  shape,  and  variations  in  these.  In  many 
of    the    corpuscles,    particularly    if    pressure    has    been 


CREMATION  45 

applied  to  the  cover-glass,  clear,  transparent  spaces, 
vacuoles,  which  may  be  either  circular,  oval,  or  even 
slit-shaped,  will  be  found  (fig.  19  c).  These  must  be 
recognized  for  what  they  are,  and  not  confounded  with 
unpigmented  parasites  nor  with  the  natural  deficiency 
in  colour  seen  towards  the  centre  of  the  corpuscles. 
From  both  of  these  the  vacuoles  can  be  distinguished 
by  the  sharpness  of  their  outline.  An  oscillatory  or 
vibratory  motion  of  the  haemoglobin  edge  of  the  vacuole 
is  highly  characteristic,  but  must  not  be  mistaken  for 
amoeboid  movement. 


Fig.   19. — a,  b,  Crenated  corpuscles  ;  c,  vacuolated  corpuscle  ;  d,  e,  buckled 

corpuscles. 


Crenation. — If  the  blood  corpuscles  be  watched  for 
some  time  they  will  be  seen  to  become  distorted  and 
projections  are  thrown  out,  either  as  a  few  blunt  pro- 
cesses or  as  sharper  projections  (fig.  19  a,  b).  This  change 
is  known  as  crenation,  and  the  projection  may  be  feebly 
motile  and  portions  may  break  off  and  be  discharged  into 
the  plasma.  These  crenations  are  readily  recognized  when 
they  occur  at  the  edge  of  the  corpuscles.  When  they 
occur  on  the  flat  surface  they  produce  an  irregularity  in 
the  colouring  and,  as  they  are  not  flat,  cause  refraction, 
and  so  produce  an  appearance  of  dark  spots  surrounded 


4''  LEUCOCYTES 

by  a  lighter  ring,  or  a  light  spot  surrounded  by  a  dark 
ring,  according  to  the  focussing. 

Some  of  the  red  corpuscles,  particularly  if  pressure  has 
been  used  in  making  the  film,  are  bent  on  themselves  or 
"buckled"  (tig.  19,  d,  e).  Such  corpuscles  may  assume 
very  varied  shapes,  and,  as  the  haemoglobin  is  readily 
expressed  from  any  part  of  the  corpuscle  irregularities  in 
colour  are  usual. 

All  these  appearances  can  be  easily  distinguished  from 
parasites  by  careful  focussing.  Occasionally,  however, 
one  meets  with  red  blood  cells  within  which  are  bodies 
closely  simulating  ring  forms.  These  may  be  motionless, 
or  have  a  slight  oscillatory  movement  at  the  edge,  but 
true  amoeboid  movement  is  not  seen.  In  shape  they 
may  be  rounded  or  oval,  and  not  uncommonly  appear 
to  possess  a  central  dark  dot,  which  is  probably  due 
to  refraction.  Similar  bodies  described  by  Cropper  and 
noted  by  many  observers  in  working  with  fresh 
blood  have  a  marked  rotary  movement.  They  are  found 
in  other  animals  besides  man,  and  in  man  and  animals 
in  non-tropical  countries.  They  have  never  been  stained 
in  dried  preparations  and  their  precise  nature  has  not 
been  determined,  but  they  are  most  frequently  found  in 
cases  where  there  is  evidence  of  degenerative  changes  in 
the  blood. 

Leucocytes  are  distinguished  by  their  size  and  the 
absence  of  colour.  As  seen  in  the  fresh  blood  they  are 
usually  granular,  the  granules  being  best  seen  on  closing 
the  iris  diaphragm  of  the  microscope.  Variation  in  the 
granules  will  be  noted,  and  the  coarse,  highly  refracting 
granules  of  the  eosinophile  leucocytes  are  quite  charac- 
teristic. These  granules  are  often  mistaken  for  pigment 
bv  beginners.  Letting  in  more  light,  which  brings  out 
pigment  granules  more  strongly  and  shows  these  normal 
granules  to  be  translucent,  will  remove  this  difficulty. 
The  characters  of  the  nuclei  and  of  the  granules  are  best 
studied  in  stained  specimens.  The  amoeboid  movements 
and  phagocytic  properties  are  best  seen  in  these  fresh 
living  fluid  films. 


STAINING   OF   FRESH    FILMS  47 

The  Blood  Platelets  are  the  most  difficult  objects 
to  see,  as  they  are  colourless,  non-granular  and  differ 
little  in  refractive  index  from  the  plasma. 

The  size  and  arrangement  in  groups,  points  that  vary- 
in  different  specimens  of  blood,  should  be  noted.  The 
irregular  serrated  margins  they  acquire  in  a  short  time, 
from  the  formation  of  filaments  of  fibrin,  are  character- 
istic of  these  bodies.  These  elements  are  more  readily 
seen  in  stained  or  over-stained  specimens. 

Staining  of  Fresh  Films. — Many  methods  of  staining 
blood,  whilst  still  in  a  fluid  condition,  by  admixture  with 
stains  have  been  employed. 

The  usual  practice  is  to  place  a  drop  of  sufficiently 
dilute  stain  on  the  slide,  then  take  a  minute  drop  of  blood 
on  the  cover-glass  and  drop  this  on  the  drop  of  stain,  so 
that  the  blood  and  stain  spread  out  together.  A  certain 
admixture  takes  place  at  the  edge  of  the  drop  of  blood, 
and  in  a  little  time  the  stain  diffuses  further  into  the 
blood. 

Various  solutions  of  stain  have  been  used.  Braddon's 
is  perhaps  as  good  as  any.*  In  this,  as  well  as  in  other 
aqueous  stains,  the  water  causes  a  liberation  of  the 
haemoglobin,  and  the  dissolved  haemoglobin  precipitates 
the  stain,  or  debris  is  stained  by  the  stain. 

If  this  process  takes  place  in  the  serum  little  confusion 
is  caused,  but  if,  as  frequently  happens,  it  takes  place 
as  the  stain  penetrates  the  red  corpuscles  it  causes  the 
formation  of  a  complicated  arrangement  of  stain  in  the 
interior  of  the  red  corpuscles,  which  has  been  mistaken 
for  parasitic  growth.  To  avoid  this  error,  a  strong  salt 
solution  is  used  by  some,  so  that  the  haemoglobin  is  not 
discharged  from  the  red  corpuscles.  Others,  for  the  same 
reason,  use  ascitic  fluid. 

Malaria  parasites  are  well  stained  by  this  method,  and 

*  Braddon's  solution  is  composed  of  1  per  cent.  pot.  citrate,  h  to  2 
per  cent,  methylene  blue  :  water  to  100  parts. 


48  DRIED    FILMS 

it  has  the  advantage  of  requiring  no  fixation,  and  conse- 
quently is  rapid. 

Dried    Films. — These  can  be  made  in   many  ways, 

most  of  which  after  a  little  practice  give  excellent  results. 
In  all  methods  it  is  important  that  only  the  top  of  the 
drop  of  blood  and  not  the  skin  should  be  touched  by 
the  slide,  cover-glass  or  paper.  Neglect  of  this  precau- 
tion will  result  in  the  admixture  of  epithelial  scales,  oil 
globules  and  micrococci  with  the  film. 

(i)  In  this  method  a  drop  of  blood  is  taken  on  the 
surface  of  a  slide  near  one  end.  The  edge  of  another 
slide  is  brought  into  contact  with  this  drop,  which  then 
spreads  out  so  as  to  fill  the  angle  between  the  two  slides 
along  the  whole  extent  of  the  line  of  contact.  On 
pushing  the  upper  slide  towards  the  other  end  of  the 
lower  slide  a  film  of  blood  will  be  left  behind.  The 
thickness  of  this  film  is  easily  regulated,  as  if  the  angle 
between  the  two  slides  is  acute  the  film  left  behind  will 
be  very  thin  ;  if  the  angle  be  nearly  a  right  angle  a  thick 
film  will  be  left.  An  angle  of  about  450  gives  the  desired 
thickness,  but  it  is  well  to  slightly  vary  the  thickness  of 
the  film  by  alternately  slightly  increasing  and  diminishing 
the  angle  made  between  the  two  slides  as  the  upper  one- 
is  pushed  along,  so  that  different  parts  of  the  film  will  be 
suitable  for  examination  for  different  purpose-. 

A  slight  modification  of  the  method  is  to  take  up  the 
drop  of  blood  on  the  edge  of  the  upper  slide  and  bring 
the  drop  of  blood  and  the  edge  of  this  slide  into  contact 
with  the  upper  surface  of  the  lower  slide  and  proceed  as 
above  (fig.  20). 

(2)  A  drop  of  blood  is  taken  on  a  slide  rather  nearer 
one  end  than  the  other,  and  the  larger  the  chop  the 
farther  from  the  middle.  Another  slide,  a  glass  rod,  or, 
perhaps  best,  the  shaft  of  a  needle,  is  then  applied  to  tin- 
drop  so  that  the  blood  spreads  along  the  whole  of  the 
line  of  contact.  The  upper  slide,  glass  rod,  or  needle  is 
then  drawn  across  the  lower  slide  and  an  excellent  film 
will  be  left  (fig.  21). 


DRIED    FILMS 


49 


Fig.    20. 


Fig.    2i. 


5° 


DKMKI)    FILMS 


(3)  Cigarette  paper,  or  gutta-percha  tissue  cut  in  the 
form  of  a  narrow  slip,  is  used  in  this  method.  The  lower 
surface  of  the  slip  is  brought  into  contact  with  the  drop 
of  blood  on  the  finger  or  ear.  This  drop  adheres  to  the 
slip  on  removal.  The  edge  of  the  slip  is  placed  on  the 
slide  and  the  blood  then  spreads  out  between  it  and  the 
tissue  paper  or  gutta-percha  tissue,  and  on  pulling  the 
tree  end  of  the  slip  a  good  but  usually  scratchy  film  will 
be  left  (fig.  22). 


Fig.   22. 


Fit; 


These  three  methods  can  be  used  for  cover-glass  pre- 
parations, particularly  if  long  cover-glasses  be  used,  but 
are  best  for  slides. 

(4)  The  last  method  advocated  is  not  satisfactory  with 
slides  but  is  useful  for  cover-glass  preparations. 

A  small  drop  of  blood  is  taken  on  one  cover-glass  and 
this  cover-glass  is  then  applied  to  a  second,  so  that  the 
blood  spreads  out  between   them.     The  cover-glasses  are 


FIXATION  51 

arranged  diagonally,  so  that  the  corners  of  each  cover- 
glass  can  be  taken  hold  of.  The  upper  cover-glass  is 
then  drawn  or  slid  over  the  lower,  care  being  taken  that 
it  is  not  lifted  off.  A  good  film  should  be  left  on  each 
cover-glass  (fig.  23). 

In  case  of  emergency  any  piece  of  flat  glass,  broken 
window-pane,  &c,  can  be  used  and  good  films  obtained, 
but  the  best  films  are  those  in  which  the  slides  are  of 
good  quality,  even  in  thickness  and  free  from  scratches, 
dirt,  grease,  or  irregularities. 

The  films,  however  made,  should  be  dried  rapidly  by 
waving  them  to  and  fro  in  the  air,  but  not  heated  ;  other- 
wise crenation  and  distortion  of  the  corpuscles  will  take 
place. 

Fixation.  —  If  such  films  were  placed  in  water  or 
aqueous  solutions  of  stains  the  haemoglobin  would  be 
dissolved  out,  and  the  corpuscles  more  or  less  destroyed  ; 
it  is  therefore  necessary  to  fix  the  films. 

Films  can  be  fixed  by  heat,  but  a  temperature  above 
a  certain  point  vacuolates  and  distorts  the  red  corpuscles. 

As  a  general  rule  in  blood  work,  fixation  bv  heat  should 
be  avoided,  though  for  one  method  of  staining  —  the 
Ehrlich-Biondi  —  fixation  by  heat  is  necessary.  Good 
results  are  more  difficult  to  obtain  in  the  Tropics  by  this 
method  than  in  England,  and,  as  the  same  information 
can  be  obtained  by  easier  methods,  it  is  not  recommended 
for  tropical  work. 

Fixation  by  absolute  alcohol,  or  by  absolute  alcohol 
and  ether  in  equal  proportions,  gives  good  and  reliable 
results.     Fix  for  ten  minutes  or  more  and  then  dry  in  air. 

There  are  other  methods  of  fixation,  and  of  these  ex- 
posure of  the  film  to  the  vapour  of  40  per  cent,  formalde- 
hyde (formalin)  for  two  minutes  is  perhaps  the  best. 

Saturated  solution  of  perchloride  of  mercury  does  not 
give  good  results  with  films  of  malarial  blood,  as  the 
parasites  do  not  stain  well  after  the  use  of  this  reagent, 
but  for  other  blood  work  the  results  are  fairly  satisfactory. 

Staining  of  Dried  Films. — When  fixed  and  dried  the  film 


52  STAINING   OF   FILMS 

can  be  stained,  and  the  number  of  stains  that  have  been 
employed  is  very  large.  Of  the  methods  most  generally 
applicable,  the  following  have  the  special  advantages  and 

drawbacks  indicated.  Often  two  or  more  methods  can 
be  employed  with  advantage  on  different  slides,  in  order 
to  bring  out  special  features  in  the  blood. 

HEMATOXYLIN.  —  Any  good  hematoxylin  stain  will 
stain  most  of  the  basic  elements  in  the  blood  and  most 
of  the  parasites.  The  number  of  preparations  used  i^ 
large.  The  formula  recommended  is  composed  of  a 
mixture  of — 

Haematin...  ...  ...  ...  2*5  grm. 

Absolute  alcohol  ...  ...         50  c.c. 

Alum        ...  ...  ...  ...  50  grm.  or  to 

saturation. 

Water        ...         ...  ...         ...    1,000  c.c. 

The  haematin  is  dissolved  in  the  alcohol  and  added  to 
the  solution  of  alum  in  water  and  the  vessel  containing 
the  mixture  is  left  loosely  corked  and  exposed  to  the  light 
in  order  to  hasten  the  maturing  of  the  stain. 

In  warm  weather  this  stain  matures  rapidly,  two  or 
three  weeks  being  sufficient.  When  mature  the  sides  of 
the  vessel  containing  the  mixture  are  deeply  stained. 
Like  all  other  hematoxylin  stains,  it  must  be  tested 
before  use  to  find  the  time  required  for  staining.  When 
properly  mature  this  preparation  requires  about  seven  to 
ten  minutes  to  stain  blood  well.  It  need  not  be  filtered 
immediately  before  use.  The  stain  may  be  placed  over 
the  film,  or  the  slide  with  the  film  on  it  may  be  immersed 
in  a  pot  of  the  stain,  which  should  be  well  shaken  before 
use. 

If  the  stain  is  placed  on  the  slide  do  not  pour  off  the 
stain,  but  flush  it  off.  If  well  flushed,  even  when  a  dirty 
stain  is  used,  little  deposit  will  be  left  on  the  film.  If  the 
stain  be  poured  off,  however  much  the  slide  is  then 
flushed  or  washed,  dirt  from  the  stain  will  adhere  to  the 
film.  After  flushing  off  the  stain  leave  in  ordinary  tap 
water  for  five  minutes.     The  effect  of  the  tap  water  is  to 


RED   CORPUSCLES  53 

change  the  dirty  purple  colour  of  the  film  to  a  clear  blue. 
The  process  is  commonly  spoken  of  as  "blueing"  the 
film.     Drain  and  allow  to  dry. 

As  a  counter-stain  eosin  is  useful.  An  aqueous  i  per 
cent,  solution  of  yellow  eosin  (soluble  in  alcohol)  is  used. 
It  will  stain  in  twenty  to  thirty  seconds ;  then  wash  and 
allow  to  dry.  A  film  so  prepared  is  in  a  fit  condition  for 
examination  with  an  oil  immersion  lens.  The  oil  can  be 
placed  directly  on  the  film,  but  if  it  is  intended  to  keep 
the  film,  it  is  simpler  to  mount  in  xylol  balsam  and  then 
examine.  Plate  IV.  shows  the  appearances  of  the  blood- 
cells  stained  in  this  manner. 

Red  Corpuscles. — The  red  corpuscles  are  stained  by 
the  eosin,  the  depth  of  the  colour  varying  according  to 
the  richness  of  the  corpuscles  in  haemoglobin.  As  in 
fresh  blood,  the  size,  depth  of  colour  and  shape  of  the 
red  corpuscle  should  be  observed.  Among  the  rarer 
forms  in  normal  blood  are  red  corpuscles  which  hardly 
stain  with  eosin,  the  shadow  or  ghost  corpuscles  ;  poly- 
chromatic corpuscles,  which  are  faintly  stained  with  both 
stains,  so  as  to  have  a  purplish  colour,  and  red 
corpuscles  containing  granules  which  stain  deeply  with 
the  basic  stain  used,  haematoxylin.  The  last-named  cells 
are  described  as  containing  basophilic  granules  (Plate 
IV.,  9).  Nucleated  red  corpuscles  are  very  rarely  present 
in  the  blood  of  healthy  individuals,  but  are  found 
not  only  in  blood  of  patients  markedly  anaemic  but  also 
in  some  cases  of  malaria,  &c.  The  nucleated  red  cor- 
puscles have  a  nucleus  frequently  fragmented  and  stain- 
ing deeply,  but  not  evenly,  with  haematoxylin  (Plate 
IV.,  3  and  4).  They  have  a  sharply  defined  margin.  Not 
unfrequently  the  nucleated  red  corpuscle  itself  is  poly- 
chromatic, or  contains  basophilic  granules.  Such  nu- 
cleated red  corpuscles  may  be  larger  (megaloblasts), 
smaller  (microblasts),  or  the  same  size  (normoblasts),  as 
the  normal. 

The  Blood  Platelets  are  stained  feebly  with  both 
stains  and  have  a  uniform  faint  purple  colour.      In  an 


54 


Will  TIC   CORPUSCLES 


overstained  specimen  the  network  of  fibrin  filaments 
starting  from  either  ;i  single  plate  or  a  group,  is  plainly 
brought  out,  but  in  a  normally  stained  specimen  only  the 
platelets  and  the  bases  of  these  filaments  are  revealed 
(Plate  IV.,  5). 


Fig.  24. — a,  Lymphocytes  ;  /',  large  mononuclear  leucocytes  ;  r,  transitional 
leucocyte  :  d,  polymorphonuclear  leucocytes;  c,  eosinophile  leucocytes. 

White  Corpuscles. — The  leucocytes  have  their  nuclei 
stained  deep  blue.  The  protoplasm  is  stained  differently 
in  the  different  varieties  of  leucocytes,  but  granules,  with 
the  exception  of  those  staining  deeply  with  eosin,  are 
not  brought  out  by  this  method.  They  are,  however, 
visible  in  the  unstained  leucocytes  and  can  be  demon- 
strated by  other  stains. 


WHITE    CORPUSCLES  5  5 

In  normal  blood  four  varieties  of  white  corpuscles  can 
be  differentiated.  Of  these  two  have  a  single,  more  or 
less  rounded,  nucleus.  These  mononuclear  leucocytes 
are  of  two  classes,  though  it  is  not  always  easy  to  say 
to  which  class  a  given  mononuclear  leucocyte  belongs. 
Still,  with  practice  the  number  of  doubtful  instances 
greatly  diminishes.  The  points  to  be  considered  in  the 
differentiation  are  the  size  of  the  corpuscle,  the  shape 
and  staining  reactions  of  the  nucleus,  the  stain  taken  up 
by  the  protoplasm,  and  the  relative  amount  of  protoplasm 
as  compared  with  the  nucleus. 

(i)  The  small  mononuclear  leucocyte,  or  lymphocyte, 
is  usually  not  much  larger  than  a  red  corpuscle  and  varies 
from  7  [x  to  12  /j,  in  diameter.  The  nucleus  stains  deeply 
and  forms  the  greater  part  of  the  corpuscle.  The  proto- 
plasm is  often  reduced  to  a  mere  rim,  and  in  any  case  is 
relatively  scanty  in  proportion  to  the  size  of  the  nucleus 
(fig.  24,  a).  The  protoplasm  is  stained  faintly  pink,  much 
the  same  as  the  protoplasm  in  the  polymorphonuclear 
leucocyte. 

(2)  The  Large  Mononuclear  Leucocytes  (fig.  24,  />), 
s*ometimes  called  the  hyaline  cells,  are  variable  in  size, 
but  some  of  them  form  the  largest  white  elements  in 
normal  blood.  The  nucleus  is  not  so  deeply  stained 
as  in  the  lymphocyte.  The  protoplasm  is  relatively 
abundant  and  stains  slightly  with  basic  stains.  It  may 
be  unstained  or  faintly  blue,  or,  if  pink,  is  less  so  than 
the  polymorphonuclear  leucocyte. 

All  these  points  have  to  be  taken  into  account  in  the 
separation  of  these  leucocytes. 

Some  corpuscles  are  found  with  the  nuclei  deeply  in- 
dented, or  horse-shoe  shaped.  In  staining  reactions  thev 
resemble  the  large  mononuclear  and  are  probably  ad- 
vanced forms  of  these,  and  not,  as  usually  described, 
transitional  forms  between  these  and  the  polymorpho- 
nuclear leucocytes  (fig.  24,  c). 

The  other  two  classes  of  leucocytes  are  much  ensier  to 
distinguish. 


56  WHITE   CORPUSCLES 

(3)  The  Polymorphonuclear  Leucocytes  (fig.  24,  d), 
sometimes  incorrectly  called  polynuclear,  form  the  greater 
number  of  the  leucocytes.  They  are  rounded  cells,  which 
are  granular  in  the  fresh  blood,  but  the  granules  arc  not 
stained  by  the  method  we  are  now  discussing.  The 
characteristic  of  these  cells  is  the  variety  in  form  of  the 
nucleus.  The  nucleus  stains  deeply  with  the  hema- 
toxylin, and  at  first  sight  appears  to  be  multiple.  Closer 
examination  shows  that  the  different  parts  of  the  nucleus 
are  really  connected  together,  though  often  by  a  mere 
string  or  filament. 

The  form  in  dried  uncompressed  specimens  is  round, 
the  size  fairly  uniform,  and  the  protoplasm  stains  a  faint 
pink. 

(4)  The  Eosinophile  Leucocytes.  —  The  fourth 
variety  has  a  deep  indented  nucleus,  sometimes  divided 
into  three.  The  nucleus  does  not  stain  so  deeply  with 
hematoxylin  as  in  the  polymorphonuclear  leucocytes, 
but  the  characteristic  of  this  leucocyte  is  the  presence  of 
a  large  number  of  coarse  granules  which  stain  deeply 
with  eosin.  Hence  these  leucocytes  are  called  eosinophile 
This  leucocyte  is  more  loosely  held  together  than 
any  other,  and  it  is  no  uncommon  event  for  one  to  be 
ruptured  in  making  the  film,  so  that  the  nucleus  is  seen 
surrounded  by   a    cloud    of   granules  stained  with  eosin 

(ng-  24>  0- 

Relative  Proportions. — These  four  varieties  of  leucocytes 

are  all  present  in  normal  blood,  but  in  relative  numbers 

varying  within  comparatively  small  limits.     The  variations 

in  appearance  are  shown  in  fig.  24. 

The  normal  proportions  are  given  variously  as  : — 

Lymphocytes      ...  ...  ...  10-25  per  cent. 

Large  mononuclear  ...  ...  5-10       ,, 

Polymorphonuclear  ...  ...  65-75        ,, 

Eosinophils       ...  ...  ...  2-4  „ 

It  will  be  seen  that  the  lymphocytes  are  the  most 
variable  elements  and,  in  an   individual,  may  vary  during 


DIFFERENTIAL   COUNTS  57 

the  same  day  from  hour  to  hour,  according  to  the  stage 
of  digestion. 

In  many  diseases,  and  for  some  time  after  these 
diseases,  there  is  a  marked  variation  in  the  relative  pro- 
portions of  these  blood  elements.  A  most  important 
variation  is  that  which  occurs  during,  and  still  more 
markedly  after,  a  malarial  attack.  The  leucocytic  varia- 
tion, which  occurs  in  malaria,  is  a  relative  increase  in  the 
number  of  large  mononuclear  elements,  so  that  they  may 
constitute  20  per  cent,  or  more  of  the  leucocytes  found- 
The  increase  appears  to  be  constant  and  it  is  rarely  less 
than  15  per  cent.  It  may  be  much  greater,  so  that  they 
may  constitute  40  per  cent,  or  even  more  of  the  total 
leucocytes. 

It  occurs  in  all  forms  of  malaria  and  persists  after  all 
other  signs  or  symptoms  of  malaria  have  disappeared. 
It  is  found  sometimes  three  months  or  more  after  an 
attack  and  rarely  disappears,  or  even  diminishes,  in  a 
month.     It  is  not  affected  by  quinine. 

A  similar  leucocytic  variation  is  occasionally,  but  rarely, 
found  in  typhoid  and  Malta  fever  and  in  other  conditions, 
but  in  these  diseases  it  is  not  persistent,  and  in  these  cases 
the  difficulty  of  distinguishing  the  true  large  mononuclear 
cells  from  the  numerous  large  lymphocytes  is  great. }  LIn 
trypanosomiasis  and  kala-azar  it  appears  to  be  constant, 
but  is  less  marked  than  in  malaria  and  is  associated  with 
an  increase  in  the  lymphocytes. 

The  total  number  of  leucocytes  varies  during  an  attack 
of  malaria.  During  the  pyrexial  period  there  is  leucopenia 
sometimes  as  low  as  2 — 3,000  per  c.mm.,  in  the  apyrexial 
periods  there  is  an  increase  to,  or  even  decidedly  above, 
the  normal.  According  to  Vincent  there  is  a  period 
immediately  after  the  paroxysm  begins  during  which 
there  is  an  increase  in  the  number  of  leucocytes,  whilst 
according  to  Ross  the  total  mononuclear  cells  inerease 
rapidly  after  a  paroxysm,  so  that  about  seven  days  later 
the  total  leucocytes  are  above  the  normal. 


58  DIFFERENTIAL   COUNTS 

A  relative  increase  in  the  polymorphonuclear  elements 
occurs  in  pneumonia  and  in  many  septic  conditions, 
particularly  when  deep-seated  abscesses  form,  such  as 
in  perityphlitic  or  hepatic  abscess.  In  such  cases  tlicrc 
is  also  an  absolute  increase  in  the  number  of  leucocytes. 
A  differential  leucocyte  count  is  therefore  an  important 
aid  in  distinguishing  these  diseases  from  malaria,  which 
they  may  resemble  clinically. 

Increase  in  the  relative  proportion  of  eosinophiles 
occurs  from  many  causes,  some  of  which  are  unknown. 
It  is  marked  in  most  cases  of  anaemia  from  ankylo- 
stomiasis, and  occurs  also  in  many  cases  of  filariasis  and 
in  some  cases  of  bilharzia  infection,  and  is  said  to  be 
well  marked  and  constant  in  trichinosis.  The  blood 
examination  may  often  give  a  hint  as  to  the  presence  of 
some  of  these  parasites.  As  the  increase  occurs  also  from 
unknown  causes,  and  in  some  skin  diseases,  and  is  often 
associated  with  chronic  bronchitis  and  asthma,  in  itself 
it  is  of  no  certain  diagnostic  value. 

The  proportion  of  lymphocytes  is  so  variable  that  only 
an  enormous  increase  is  of  importance.  This  occurs  in 
some  cases  of  scurvv  and  is  associated  with  an  increase 
in  the  other  mononuclear  elements. 

A  differential  leucocyte  count  must  not  be  confused 
with  an  actual  enumeration  of  the  number  of  leucocytes 
present  in  a  given  volume  of  blood.  That  has  to  be 
determined  separately,  as  we  shall  see  subsequently.  To 
make  a  differential  count  of  the  leucocytes  a  dried  him 
of  a  small  but  uncertain  volume  of  blood  is  prepared  and 
stained.  All  the  leucocytes  found  in  a  systematic 
examination  of  a  part  of  this  film  are  counted  and  the 
percentage  of  each  different  variety  met  with  is  thus 
ascertained.  For  accurate  work  not  less  than  500  should 
be  counted,  but  for  clinical  purposes  200  will  often 
suffice.  The  edges  of  the  film  where  leucocytes  are  most 
numerous  should  not  be  included  in  the  enumeration. 

Variations  in  the  total  number  of  leucocytes  and  in  the 
relative  proportion    of  the   different   kinds   of    leucocytes 


DIFFERENTIAL    COUNTS 


59 


occur  in  healthy  persons  to  a  moderate  extent.  The 
number  of  leucocytes  in  healthy  adults  is  rarely  under 
6,000  per  c.mm.  or  over  12,000.  In  new-born  children 
the  number  is  much  greater — up  to  20,000,  and  in  preg- 
nancy is  increased  up  to  15,000.  During  active  digestion 
there  is  an  increase  in  the  number  of  leucocytes. 

Variations  in  the  relative  proportion  of  the  different 
forms  of  leucocytes  readily  occur  in  healthy  children  : 
but  in  adults,  with  the  exception  of  the  lymphocytes, 
such  variations  are  comparatively  small. 

The  following  table  gives  examples  of  the  relative  pro- 
portions of  the  different  leucocytes  in  certain  diseases, 
as  well  as  the  number  of  leucocytes  per  c.mm.  usually 
met  with  in  such  diseases  :■ — 


No.  per  c.mm. 

Poly- 
morpho- 
nuclear 

Lympho- 
cytes 
Per  cent. 

Large 
mono- 
nuclear 
Per  cent. 

Eosino- 

philes 

Per  cent. 

Per  cent. 

Pneumonia    ... 

Great  increase  up  to 

60,000 

85  to  95 

15 

5 

I 

Sepsis... 

Increase  up  to  30,000 
or  40,000 

75  to  90 

15  to  25 

5  to  10 

i  to  8 

Liver  abscess... 

Increase  varies,  often 
slight,       12,000     to 
20,000 

75  to  85 

15  to  25 

5  to  10 

2  to  4 

Typhoid 

Slight  increase  at  most 

50  to  65 

25  to  40 

5  to  15 

1  to  3 

Malta  fever    ... 

>>           j  >           ?> 

50  to  65 

25  to  40 

5  to  15 

1  to  3 

Relapsing  fever 

Great  increase  up  to 
50,000 

75  to  90 

10  to  20 

5  to  10 

1  to  2 

Malaria 

No  increase;  decrease 
during  pyrexia 

45  to  65 

15  to  25 

15  to  30 

I 

Trypanosomia- 
sis 
Kala  azar 

No  increase  ... 

50  to  65 

20  to  30 

15  to  20 

2  to  4 

Marked     decrease  — 

50  to  60 

25  to  35 

15  to  20 

2  to  4 

1,000  to  3,000 

Ankylostomia- 

Usually increased,  es- 

66 to  70 

10  to  20 

5  to  10 

8  to  50 

sis 

pecially  in  early  cases 

Beri-beri 

Slight     increase  — 
11,000  to  14,000 

24  to  49 

30  to  68 

I   to   12 

1  to  5 

Pellagra 

7,000  to  9,000 

50  to  60 

32  to  42 

3  to  6 

I  to  4 

It  must  be  remembered  that  multiple  infections  are 
common.  In  relapsing  fever  lung  complications  are  so 
common  that   possibly  the    leucocytosis    is    due   to  this. 


MYELOCYTES 

'he  tendency  to  leucocvtosis  and'an  increase  in  the  poly- 
morphonuclear leucocytes  due  to  any  disease  will  mask 
the  mononuclear  increase  due  to  malaria. 

Abnormal  Cells.  —  Abnormal  elements  resembling 
leucocytes  are  present  in  certain  diseases,  particularly 
in  leucocythaemia.  These  abnormal  elements  are  known 
as  Myelocytes  (fig.  25)  from  their  similarity  to  cells 
found  normally  in  the  bone-marrow.  They  are  of  three 
kinds,  all  mononuclear. 

(1)  The  first  form  is  variable  in  size,  the  greater 
number  of  them  being  much  larger  than  the  large  mono- 
nuclear leucocytes.  With  eosin  and  hematoxylin,  as 
the  granules  which  they  contain  are  not  stained,  it  is 
sometimes  difficult  to  distinguish  the  smaller  ones  from 
the  larger  of  the  mononuclear  leucocytes.  For  practical 
purposes  the  difficulty  is  unimportant,  as  when  myelo- 
cytes occur  they  are  common  and  most  of  them  are 
readily  distinguished  from  the  leucocytes. 


In  these  myelocytes  the  nucleus  stains  less  readily  and 
is  therefore  paler  than  that  of  the  large  mononuclear 
leucocytes.  The  edge  of  the  nucleus  is  frequently  ragged. 
The  protoplasm  is  abundant  and  stains  in  many  cases 
more  deeply  with  eosin  than  the  large  mononuclear 
leucocytes  do.     Mitotic  figures  are  often  met  with. 

(2  and  3)  The  other  two  forms  of  myelocytes  contain 
granules  which  stain  deeply  with  eosin.  They  are  sub- 
divided according  to  the  size  of  these  granules,  which 
may  be  coarse,  as  in  the  eosinophile  leucocytes,  or  fine. 


MYELOCYTES  6 1 

The  distinction  is  probably  unimportant.  These  myelo- 
cytes are  distinguished  at  once  from  eosinophile  leucocytes 
by  the  single  nucleus,  and  from  each  other  by  the  size 
of  the  eosinophile  granules. 

These  cells  are  abnormal  cellular  constituents  that 
may  be  met  with  in  blood  specimens  stained  with  eosin 
and  hcematoxylin,  and  they  must  be  clearly  recognized 
before  any  satisfactory  examination  for  parasites  can 
be  made.  In  themselves  they  are  of  considerable  im- 
portance in  the  recognition  of  various  diseases  and  for 
prognosis. 

Cells  known  as  "  mast-cells "  are  occasionally  found 
in  normal  blood  and  more  abundantly  in  some  of  the 
blood-diseases,  e.g.,  leucocythsemia,  &c.  They  are  prob- 
ably degenerated  leucocytes,  usually  of  the  polymorpho- 
nuclear type.  The  protoplasm  of  the  cell  contains 
numerous  coarse  metachromatic  granules  and  the  nucleus 
is  often  obscured.  These  cells  are  best  shown  when 
stained  by  the  Romano wsky  method  or  one  of  its 
modifications. 

Their  significance  is  unknown; 

In  pernicious  anaemia  and  in  chlorosis  the  changes 
in  the  red  corpuscles,  the  irregularity  in  their  size,  shape, 
and  colouring  are  of  clinical  value,  and  in  most  tropical 
anaemias,  including  that  occurring  in  malaria,  the  changes 
are  similar  to  those  in  a  mild  case  of  pernicious 
anaemia.* 

Leucocythaemia  is  readily  recognized  by  the  enormous 
increase  in  the  number  of  the  white  elements  which,  as 
we  have  seen,  take  on  basic  stains.  This  increase  is  so 
great  that  the   appearance    of   a  dried    film    indicates    it 

*  Between  these  two  forms  of  anaemia  the  main  difference 
observed  in  blood  examination  is  that  in  chlorosis  the  number  of 
corpuscles  is  not  diminished,  but  the  haemoglobin  is,  so  that  each 
corpuscle  is  poor  in  haemoglobin.  In  pernicious  anaemia  there  is 
a  great  diminution  in  the  number  of  corpuscles,  but  the  haemoglobin 
value  of  the  corpuscles  averages  much  the  same  as  normal  blood. 
Mixed  or  intermediate  cases  occur. 


02  DOUBLE   STAINS 

unmistakably,  and  it  is  not,  for  diagnostic  purposes, 
necessary  to  make  any  count.  The  presence  in  numbers 
of  the  eosinophile  myelocytes  is  conclusive  proof  of  the 
implication  of  the  bone-marrow,  whilst  the  absence  of 
this  form  of  abnormal  cell  indicates  more  probably  a 
lymphatic  leucocytha?mia. 

In  all  forms  of  leucocythannia  decided  changes  are  also 
found  in  the  red  corpuscles.  Irregularities  in  size,  shape 
and  depth  of  colour  are  common,  and  nucleated  red 
blood  corpuscles  occur,  often  in  large  numbers.  Poly- 
chromatic red  corpuscles  and  red  corpuscles  showing 
basophilic  granules  are  also  common. 

If  no  abnormal  cells  are  present  the  relative  propor- 
tions of  the  normal  cells  may  be  so  changed  that  we  can 
diagnose  with  some  degree  of  probability  septic  processes, 
recent  malaria,  or  helminthiasis. 

Eosin  and  haematoxylin  can  be  used  for  staining  any  of 
the  parasites  found  in  blood.  The  stains  are  easily  prepared, 
keep  well,  and  their  use  is  not  dependent  on  distilled  water 
or  appliances  which  are  not  obtainable  everywhere. 

It  is  not  a  very  brilliant  stain,  and  therefore  other 
stains  giving  more  marked  contrast  are  for  some  purposes 
preferable.  It  does  not  stain  the  granules  present  in 
many  [of  the  white  elements  of  the  blood,  and  though 
of  general  application,  other  stains  are  of  greater  value 
for  special  purposes  and  have  special  advantages. 

Double  Stains. — Combinations  of  methylene  blue 
and  eosin  dissolved  in  methyl  alcohol  are  much  used. 
The  first  is  the  Louis  Jenner  stain.  It  is  made  by 
adding  an  aqueous  eosin  solution  to  one  of  methylene 
blue.  The  stains  combine  and  form  a  precipitate  which 
is  collected  in  a  filter,  dried  and  dissolved  in  methylic 
alcohol. 

This  stain  can  only  be  used  with  films  that  have  not 
been  fixed.  The  methylic  alcohol  does  all  the  fixing 
required.     Distilled  water  is  an  essential. 

The  stain  may  be  placed  on  the  film,  slide  or  cover- 
glass  for  three  and  a  half  to  four  minutes,  or,  and  this  is 


leishman's  stain  63 

better,  the  slide  or  cover-glass  can  be  placed  in  the  stain 
in  a  well-stoppered  bottle  for  the  same  length  of  time. 
The  time  must  be  kept  accurately,  carelessness  in  this 
respect  leading  to  poor  results.  The  stain  must  be 
flushed  off  with  distilled  water,  and  it  is  better  to  allow 
the  distilled  water  to  stand  on  the  film  for  half  a  minute 
after  washing.  The  water  can  then  be  drained  or  blotted 
off,  the  film  allowed  to  dry,  and  the  specimen  examined 
directly  with  the  oil  immersion.  When  it  is  considered 
desirable  to  keep  the  specimen  a  drop  of  xylol  balsam 
should  be  placed  on  the  film  and  covered  with  a  cover- 
glass. 

With  this  stain  the  red  blood  corpuscles  are  stained 
pink,  the  depth  of  colour  varying  with  the  amount  of 
haemoglobin  which  the  corpuscles  contain. 

The  nuclei  of  the  leucocytes  are  stained  a  clear  blue, 
the  eosinophile  granules  are  stained  deep  red,  and  the 
granules  in  the  polymorphonuclear  leucocytes,  which  it 
will  be  remembered  are  not  stained  with  eosin  and 
hematoxylin,  are  brought  out  as  fine  dull-reddish 
granules. 

Basophilic  granules  contained  in  cells  are  stained  blue ; 
this  occurs  both  in  white  cells  and  in  some  red  corpuscles. 
This  stain  is  a  good  stain  for  many  parasites,  particularly 
those  of  malaria.     Bacilli  and  cocci  are  also  stained  blue. 

Some  specimens  of  the  Louis  Jenner  stain  bring  out 
clearly  the  important  constituent  of  nuclei  known  as 
chromatin.  A  modification  of  the  method  and  of  the 
methylene  blue  is  required  to  bring  out  the  chromatin 
with  certainty.  Many  methods  have  been  employed  for 
this  purpose,  most  of  them  modifications  of  Romanowsky's 
method. 

Leishman's  Stain. — The  simplest,  the  most  rapid,  and 
on  the  whole  the  most  satisfactory  of  these  methods  is 
that  introduced  by  Leishman.  A  saturated  aqueous 
solution  of  methylene  blue,  preferably  "  Hochst's  pure 
medicinal,"  is  made.  This  solution  has  to  be  rendered 
polychrome,  so  that  in  addition  to  the  pure  blue  colour 


64  LEISHMAN'S   STAIN 

of  the  ordinary  methylene  blue  it  is  in  part  changed  into 
a  red  stain.  The  change  is  indicated  by  a  change  in 
colour  of  the  solution,  so  that  in  thin  layers  it  has  a 
reddish  tinge.  The  solution  of  methylene  blue  becomes 
to  some  extent  polychrome  when  exposed  to  air  for  some 
months,  but  for  practical  purposes  a  quicker  transformation 
is  required.  There  are  many  methods.  Repeated  heating 
in  a  sterilizer  accelerates  the  change.  Leishman  uses  a 
1  per  cent,  solution  of  methylene  blue  (Griibler's),  and 
adds  5  per  cent,  sodium  carbonate  to  it.  This  solution 
he  keeps  at  a  temperature  of  650  C.  for  twelve  hours,  and 
then  exposes  to  air  for  a  week  or  more. 

J.  H.  Wright  adds  to  a  h  per  cent,  solution  of  sodium 
bicarbonate  1  per  cent,  of  methylene  blue  (Griibler's 
B.  X.,  Koch's  or  Ehrlich's  rectified).  This  solution  is 
steamed  in  a  steam  sterilizer  for  one  hour  to  effect 
the  required  transformation,  and  the  solution  may  be 
used  as  soon  as  it  is  cold  without  filtering. 

A  convenient  method,  and  more  suitable  where  steam 
sterilizers  are  not  available,  is  to  treat  the  saturated  solu- 
tion of  methylene  blue  with  freshly  precipitated  oxide 
of  silver.  A  solution  of  sodium  hydrate  is  added  to  a 
solution  of  nitrate  of  silver  till  no  more  precipitate  forms. 
The  precipitate  is  washed  till  the  washings  are  neutral 
to  litmus  paper.  The  precipitate,  oxide  of  silver,  is 
added  to  the  saturated  solution  of  methylene  blue,  and 
it  is  allowed  to  stand  for  twenty-four  hours  or  more.  A 
considerable  proportion  of  the  methylene  blue  in  solu- 
tion will  be  converted  into  polychrome  methylene  blue. 
The  superjacent  solution  should  be  decanted  off  from 
the  precipitated  silver  salts  and  filtered  before  use.  It 
improves  with  keeping. 

Whatever  method  be  adopted  for  rendering  the  methy- 
lene blue  polychrome  the  subsequent  proceedings  are 
the  same. 

One  hundred  cubic  centigrammes  of  this  solution 
of  polychrome  methylene  blue  are  placed  in  a  large 
shallow  vessel    (a  half-plate  photographic  tray  is  a  suit- 


leishman's  stain  65 

able  one),  and  then  a  i  in  1,000  aqueous  eosin  solution  is 
added  till  a  thick  film  forms  on  the  surface  and  the  fluid 
just  shows  the  colour  of  the  eosin.  About  400  c.c.  or  a 
little  more  will  be  required,  but  the  change  in  colour  is 
the  guide.  The  mixture  should  be  well  stirred  and  then 
allowed  to  stand  exposed  freely  to  air  for  some  hours, 
stirring  occasionally,  and  afterwards  filtered.  The  residue 
in  the  filter  is  composed  of  the  stain.  It  should  be  well 
washed  with  distilled  water  till  the  washing  has  only  a 
faint  bluish  tinge,  and  then  thoroughly  dried,  preferably 
in  an  incubator  at  blood  heat.  The  stain  must  be  finely 
powdered  before  use. 

Two  centigrammes  of  the  powder  can  then  be  dissolved 
in  100  c.c.  of  pure  methyl  alcohol,  and  the  stain  is  ready 
for  use.  It  is,  perhaps,  more  convenient  to  make  a 
saturated  solution  of  the  stain  in  methyl  alcohol,  filter 
in  the  cold,  and  dilute  with  one-tenth  of  its  bulk  of 
methyl  alcohol,  so  that  a  solution  is  made  which  is  not 
quite  saturated.  The  "  tabloids  "  of  the  stain  (Leishman's 
stain)  keep  well,  and  give  excellent  results.  It  is  cleaner 
than  the  home-made  stain,  but  the  preparations  do  not 
keep  so  well. 

To  Use  the  Stain. — With  a  pipette  two  or  three  drops 
of  the  stain  are  placed  on  the  dried  unfixed  blood-film 
on  slide  or  cover-glass,  and  allowed  to  stand  on  it  for 
half  to  one  minute.  If  it  shows  any  tendency  to  dry 
over  any  parts  of  the  film  in  this  period  fresh  stain 
must  be  added.  To  the  fluid  stain  on  the  slide  at  the 
expiration  of  this  half  or  one  minute  distilled  water 
must  be  added  drop  by  drop,  and  by  oscillating  the  slide 
the  stain  and  water  are  mixed  as  rapidly  as  possible. 
The  amount  of  water  required  should  be  about  double 
that  of  stain,  but  a  better  guide  is  to  add  the  water 
in  such  an  amount  that  when  mixed  with  the  stain  the 
dark  blue  colour  of  the  latter  is  replaced  by  a  pinkish 
colour  in  the  mixture,  whilst  the  precipitated  stain  can 
be  seen  floating  in  the  fluid.  With  a  little  practice 
the  right  amount  of  water  required  in  each  case  is  easily 

5 


66  leishman's  stain 

found,  and  slight  variations  from  exactitude  arc  not  of 
great  importance.  The  water  mixed  with  the  stain  should 
be  allowed  to  remain  on  the  him  for  five  minutes,  or  with 
old  or  thick  films  for  a  longer  period.  It  is  quite  easy  to 
watch  the  staining  under  a  low  power  on  the  microscope, 
and  the  staining  of  the  leucocytes  is  the  best  guide. 

The  stain  is  then  flushed  off  with  distilled  water,  and 
a  drop  of  distilled  water  is  ailowed  to  remain  on  the 
film  for  about  one  minute.  A  certain  amount  of  the 
blue  is  dissolved,  and  the  red  corpuscles  acquire  a  clearer 
red  colour.  This  clearing  with  distilled  water  is  essential 
to  obtain  good  results.  The  more  deeply  the  specimen 
is  stained  the  longer  will  be  the  time  required  for  clearing. 
This  stage  of  the  process  is  watched  under  the  micro- 
scope and  stopped  when  the  clearing  is  sufficient.  The 
water  is  then  washed  off  rapidly  with  distilled  water,  the 
specimen  drained  or  blotted  and  allowed  to  dry.  Mount 
in  xylol  balsam  and  examine. 

The  principle  of  all  modifications  of  the  Romanowsky 
stain  for  chromatin  is  that  the  staining  takes  place  during 
the  precipitation  of  the  stain,  in  the  original  processes 
during  the  precipitation  of  the  mixture  of  aqueous  solu- 
tions of  the  stains,  and  in  Leishman's  method  during  the 
precipitation  by  water  of  the  combined  stains  dissolved 
in  methyl  alcohol. 

Absolute  alcohol  with  2  per  cent,  aniline  oil  can  be 
used  as  the  solvent  instead  of  methyl  alcohol,  and  the 
solution  treated  as  Leishman  treats  the  methyl  alcohol 
solution.  The  results  are  not  so  good  as  with  methyl 
alcohol  for  a  solvent,  but  are  very  fair.  It  is  not  to  be 
recommended  except  where  methyl  alcohol  cannot  be 
obtained  :  the  time  for  all  the  stages  of  the  process  should 
be  doubled  if  this  solvent  be  employed. 

Other  modifications  can  be  used  for  films  previously 
fixed  in  alcohol  and  ether. 

In  the  first  of  these  staining  takes  place  during  the 
admixture  and  mutual  precipitation  of  the  eosin  and 
polychrome  methylene  blue.     A  1  per  cent,  solution  of 


LEISHMAN'S   STAIN  67 

pure  medicinal  methylene  blue  (Grubler's)  is  made  in 
distilled  water  and  ^  per  cent,  sodium  carbonate  added, 
This  solution  keeps  well  and  is  fit  for  use  when  a  reddish 
tinge  appears.  This  change  is  expedited  by  keeping  in 
an  incubator.  A  second  solution  is  a  1  in  1,000  aqueous 
solution  of  eosin  extra  B.A.  (Grubler).  This  is  fit  for 
use  at  once,  and  keeps  well  if  not  exposed  to  light. 

These  are  stock  solutions,  and  should  be  diluted  with 
twenty-four  parts  of  pure  water  before  use.  The  solutions 
are  rapidly  mixed  and  stirred,  and  the  slides  or  cover- 
glasses  are  placed  with  the  film  side  downward  in  the 
mixture.  The  dish  should  be  rocked  from  time  to  time, 
and  the  films  left  in  the  stains  for  half  an  hour  or  more 
till  well  soaked.  This  is  tested  by  examination  of  the 
slide  whilst  still  wet  under  a  low  power. 

The  specimen  should  then  be  washed  in  distilled  water, 
rapidly  dried,  and  examined  again  under  a  low  power. 
If  too  deeply  stained  a  little  distilled  water  may  be  left 
on  the  slide  for  a  minute  or  more  to  clear  it.  Blot  off 
the  water,  dry  in  the  air  and  examine  directly,  or  after 
mounting  in  Canada  balsam. 

Giemsa's  Method. — This  is  another  modification  use- 
ful for  fixed  films  where  intense  basic  staining  is  required, 
and  also  for  the  restoration  of  faded  preparations. 

The  staining  mixture  consists  of  : — 

Azur  ii.,  eosin  ...  ...  ...  -3  grm. 

Azur  ii.         ...  ...  ...  ...         -8     ,, 

Glycerine  (Merck,  chemically  pure)  250  grm. 

Methyl  alcohol  (Kahlbaum  i.)      ...  250     .,, 

Immediately  before  use  the  stain  is  diluted  with  distilled 
water,  1  of  stain  to  15  of  distilled  water. 

The  films  are  fixed  in  methylic  alcohol  for  two  to  three 
minutes,  or  in  absolute  alcohol  for  five  minutes  and  then 
dried. 

The  diluted  stain  is  poured  on  the  films,  and  left  from 
fifteen  to  thirty  minutes,  or  for  deep  staining  up  to 
twenty-four  hours. 


68  (ill-.. MSA's    .METHOD 

The  films  are  then  Hushed  with  a  strong  jet  of  tap 
water,  dried  in  the  air,  and  mounted  in  Canada  balsam. 

Eosin  Azur  Method. — This  method  is  a  modification 
of  Giemsa's  method,  and  has  the  advantage  over  the 
latter  of  being  more  rapidly  carried  out.  The  stain  is 
supplied  in  "tabloid"  form. 

To  make  the  solution  one  "  tabloid"  is  dissolved  in  10 
c.c.  of  pure  methyl  alcohol,  and  the  mixture  allowed  to 
stand  for  twenty-four  hours.  Only  unfixed  films  must  be 
used.  The  details  of  the  process  are  the  same  as  laid 
down  for  Leishman  staining,  with  the  exception  that  the 
dilution  of  the  staining  solution  with  distilled  water 
must  be  greater  :  one  part  of  stain  to  three  parts  of 
distilled  water  gives  the  best  results.  Staining  takes 
seven  to  fifteen  minutes.  This  method  is  especially  of 
advantage  in  staining  those  parasites  in  which  a 
difficulty  is  experienced  in  bringing  out  the  chromatin 
clearly  with  other  stains.  It  is  particularly  useful  for 
staining  trypanosomes  and  halteridium. 

in  specimens  of  blood  stained  by  Romanowsky's 
method  and  its  modifications,  there  are  several  distinct 
colours  to  be  observed  (Plates  V.  and  VI.). 

Chromatin  is  stained  red.  Other  elements  taking  basic 
stains  are  mostly  stained  blue  in  various  shades,  and  the 
red  corpuscles  are  stained  a  peculiar  pale  pink  with  the 
eosin.  Some  granules,  as  those  in  the  so-called  "  mast 
cells,"  are  said  to  be  metachromatic,  as,  though  they  stain 
deeply,  the  colour  is  different  to  that  of  any  of  the  com- 
ponent colours  of  the  stain  used  (Plate  V.,  7). 

Polychromatic  red  corpuscles  are  stained  purple  and 
basophilic  granules  are  well  brought  out  as  blue  dots. 
The  nuclei  of  nucleated  red  blood  corpuscles  are  found 
to  be  rich  in  chromatin,  and  consequently  the  nuclei 
are  stained  a  deep  violet-purple. 

In  corpuscles  invaded  by  certain  parasites,  viz.,  those 
of  human  benign  tertian  malaria,  granules  or  dots  staining 
red  are  found.  In  amphibian  blood  corpuscles  invaded 
by  one  species  of  haemogregarine,  similar  granules  occur. 


BLOOD    PLATELETS  69 

These  granules  are  known  as  Schiiffner's  dots,  and 
indicate  a  peculiar  form  of  degeneration  (Plate  V.,  21, 
and  Plate  VI.,  7,  8,  9). 

Granules  staining  with  the  basic  stain,  blue,  are  some- 
times seen  in  corpuscles  invaded  with  subtertian  parasites. 
They  resemble  Plehn's  bodies  but  are  somewhat  larger. 
They  have  been  called  Maurer's  bodies.  Plehn's  bodies 
or  these  bodies  are  in  some  cases  of  malaria  very  common 
in  the  uninfected  corpuscles  (Plate  VI.,  20). 

The  Blood  Platelets  are  stained  faint  blue,  with 
numerous  red  particles  which  sometimes  form  a  mesh- 
work.  These  particles  are  deeply  stained  and  render 
the  platelets  very  conspicuous  (Plate  V.,  2). 

The  Leucocytes,  with  the  exception  of  the  eosinophile, 
are  well  stained,  but  in  well-stained  specimens  the  eosino- 
phile granules  do  not  show  a  clear  red,  as  the  protoplasm  in 
which  they  are  embedded  stains  3.  deep  blue.  The  large 
size  of  the  granules  is  shown,  and  there  is  no  real  difficulty 
even  in  a  badly  stained  specimen  in  recognizing  these 
elements.  They  do  not,  however,  form  such  conspicuous 
objects  as  in  specimens  stained  by  Louis  Jenner's  stain. 

The  nuclei  of  the  polymorphonuclear  leucocytes  stain 
purple.  The  staining  is  not  regular  but  in  patches.  The 
protoplasm  contains  minute  granules,  usually  in  very 
large  numbers,  staining  brownish-red.  The  protoplasm 
itself  is  very  faintly  stained. 

The  Large  Mononuclear  Leucocytes. — The  nuclei 
stain  faintly  purple.  The  staining  is  not  uniform,  but 
usually  presents  a  faint  mottled  appearance.  The  proto- 
plasm stains  a  faint  blue,  and  imbedded  in  it  are  granules, 
which  may  be  coarse  or  fine,  and  stain  a  deep  clear  red. 
These  are  known  as  chromidia  (Plate  V.,  4). 

The  Lymphocytes. — The  nuclei  stain  a  deep  purple 
from  the  large  amount  of  chromatin  contained.  The 
staining  is  more  uniform  than  in  most  of  the  leucocytes. 
The  protoplasm  is  stained  deep  blue,  is  nearly  uniform,  and 
has  no  granules  staining  a  different  colour  (Plate  V.,  3). 

Mast  Cells. — The  nuclei  are  stained  very  faintly,  and 


70  WHITE   CELLS 

when  the  basophilic  granules  arc  numerous  are  difficult 
to  make  out.  The  granules  in  the  protoplasm  form 
large  and  irregular  masses,  and  stain  a  deep  purple- 
brown  (metachromatic)  (Plate  V.,  7). 

Myelocytes  have  in  most  cases  a  rather  feebly  stain- 
ing nucleus,  poor  in  chromatin.  The  nuclei  are  large, 
but  the  relative  amount  of  protoplasm  varies  greatly ; 
in  many  cases  a  mere  rim  only  of  protoplasm  is  found 
(Plate  V.,  10,  11,  12,  13). 

Granules  taking  either  the  acid  or  basic  stain,  or  both, 
are  present  in  most  of  the  cells,  sometimes  in  small,  but 
more  commonly  in  large  numbers.  Sometimes  two,  or 
even  three,  classes  of  granules  are  present  in  the  one  cell 
(Plates  III.,  V.,  10  to  13). 

A  detailed  classification  of  these  abnormal  cells  would 
be  very  difficult,  as  intermediate  forms  abound. 

Amongst  these  cells  are  a  small  number  with  a  large 
nucleus  richer  in  chromatin  than  most  of  the  myelocytes, 
and  a  rim  of  protoplasm  staining  a  deep  blue.  These 
are  not  unlike  the  large  cells  found  in  cases  of  trypano- 
somiasis and  in  other  ill-determined  blood  conditions, 
but  in  those  the  protoplasm  is,  relatively  to  the  nucleus, 
in  larger  amount  (Plate  V.,  9). 

The  true  myelocytes  include  the  eosinophile  myelocytes, 
but  in  these  there  is,  in  most  cases,  some  admixture  of 
neutrophile  or  basophile  granules,  as  shown  by  this  stain. 

The  main  classes  of  granules  revealed  by  Leishman's 
stain  are  pure  oxyphile  or  eosinphile  staining  pink, 
basophile  staining  blue,  and  neutrophile,  which  take  up 
both  acid  and  basic  stains,  including  in  some  instances 
the  red  modification  of  the  methylene  blue,  and  meta- 
chromatic granules.  According  to  the  relative  proportions 
of  the  three  stains,  these  granules  may  present  a  range  of 
colours  from  blue  to  red,  or  to  a  purple-brown,  and  also 
differ  in  the  intensity  of  the  staining  and  in  the  size  of  the 
granules.  Too  little  is  known  of  the  micro-chemistry  of 
such  cells,  or  of  their  origin  in  detail,  for  the  meaning 
and  value  of  the  different  granules  found  to  be  of  much 
practical  importance  at  present. 


CHAPTER    IV. 

Animal  Parasites  Found  in  Blood.  —  Of  the  four 
great  divisions  of  the  protozoa,  representatives  of  the 
sporozoa  and  mastigophora  only  are  found  in  human 
blood. 

To  the  Sporozoa  belong  the  parasites  which  cause 
malaria  in  man.  These  are  found  in  the  red  blood 
corpuscles. 

The  Mastigophora  (flagellate  organisms)  are  repre- 
sented by  trypanosomes,  and  spirochaetae,  which  are  found 
in  the  blood  plasma,  and  also  by  Leishman-Donovan 
bodies  found  in  the  leucocytes. 

Belonging  to  the  higher  animal  kingdom  are  Tre- 
MATODA,  of  which  the  Schistosomum  hcematobiuin  and 
S.  japonicuni  are  found  in  certain  blood-vessels,  and 
Nematoda,  represented  by  the  filaria  and  filarial  embryos, 
or  micro-filaria.  Sexually  mature  forms  are  found  in  the 
blood  in  lower  animals,  and  one  only,  F.  magalhaesi,  once 
in  man. 

Examination  of  the  Blood  for  Protozoa. — An 
essential  feature  of  the  examination  consists  in  the 
examination  of  the  fluid  blood  as  soon  as  possible  after 
its  removal  from  the  body.  Many  of  the  parasites  exist 
in  the  red  blood  corpuscles,  so  that  the  film  must  be 
so  thin  that  in  a  great  part  of  it  the  red  corpuscles 
are  all  lying  flat  and  separate  from  each  other.  The 
methods  of  making  such  thin  fluid  films,  already  de- 
scribed, must  be  strictly  adhered  to.  It  is  often  urged 
that  examinations  of  stained  films  are  more  convenient 
and  better,  but  it  cannot  be  insisted  upon  too  strongly 
that  most  of  the  important  errors  which  have  occurred 
have  been  due  to  the  exclusive  use  of  stained  specimens, 
and  also  that  the  phenomena  of  life  can  only  be  satis- 


72  PROTOZOA    IN    DRIED    FILMS 

factorily  observed  in  the  fluid  Mood.  These  include 
some  points  of  diagnostic  value,  namely,  the  character 
and  movements   of    pigment,   the  activity  of   amoeboid 

movement  and  the  formation   of  flagella. 

Stained  films  have  their  value,  and  show  more  clearly 
some  points  in  the  structure  of  the  parasites.  In  busy 
practice  it  is  often  more  convenient  to  defer  for  some 
hours  the  examination  of  the  films,  and  in  such  cases 
stained  specimens  are  more  useful.  In  any  case  ol 
difficulty,  or  when  dealing  with  a  parasite  believed  to  be 
new,  both  methods  should  be  employed. 

Dried  films  can  be  made  by  any  of  the  methods  already 
described,  and  the  parasites  stained  by  the  methods 
recommended.  The  films  deteriorate  when  kept,  and 
should  therefore  be  examined  as  early  as  convenient, 
though  a  delay  of  a  few  days  is  not  of  much  importance. 

Other  methods  can  be  adopted  if  only  the  presence 
or  absence  of  parasites  has  to  be  determined. 

The  methods  generally  used  can  be  divided  into  three 
groups  :  — 

A. — Those  in  which  preliminary  fixation  is  required 
before  staining. 

B. — Those     in     which    fixation    arid    staining    are 
effected  together. 

C. — Those  in  which  preliminary  fixation  is  avoided. 
A. — Films  are  fixed  by  immersion  in  absolute  alcohol 
or  in  absolute  alcohol  and  ether  for  ten  minutes  or  more. 
(i)  HEMATOXYLIN  alone,  or  HEMATOXYLIN  and  EOSIX. 
These  stains  can  be,  used  as  already  described,  but  better 
results  are  obtained  by  doubling  the  time  for  staining 
with  hematoxylin. 

(2)  Borax  Methylene  Blue. — This  stain  is  com- 
posed of  methylene  blue  2  grm.,  borax  5  grm.,  and 
water  100  c.c. 

Place  a  few  drops  of  the  stain  on  the  dried  and  fixed 
film  and  leave  it  for  thirty  seconds.  Wash  well  with 
water,  allow  to  dry,  and  examine  directly,  or  mount  in 
xylol  balsam. 


STAINING    METHODS  73 

It  is  very  easy  to  overstain  -by  this  method,  and  in  such 
a  case  the  red  corpuscles  will  also  be  stained  a  deep  blue 
and  the  parasites  will  not  stand  out  clearly. 

This  stain  is  most  rapid  in  its  action,  and  on  account 
of  the  risk  of  over-staining  some  authorities  dilute  it  with 
one,  two,  or  three  times  the  volume  of  water.  The  stain 
keeps  well. 

(3)  Carbol  Thionin. — A  stock  solution  of  thionin 
1^  grm.,  alcohol  10  c.c,  and  1  in  20  aqueous  carbolic 
acid  solution  to  100  c.c.  is  made.  This  stock  solution 
keeps  well,  but  is  too  strong  to  use  for  films.  Before 
use  it  should  be  diluted  with  ^three  parts  of  water  and 
filtered.  This  diluted  solution  does  not  keep  for  more 
than  a  few  days.  For  use,  cover  the  film  and  leave  the 
stain  on  for  five  minutes  or  more.  It  does  not  easily 
over-stain,  so  that  only  the  minimum  time  need  be 
remembered  ;  still,  to  get  good  results,  half  an  hour  is  about 
the  limit.  Flush  off  the  stain,  allow  to  dry,  and  examine 
directly,  or  mount  in  Canada  balsam.  Old  films  stain  with 
this  method  much  more  rapidly  than  ones  recently  made. 

It  is  a  good,  clear,  transparent  basic  stain  and  gives  a 
very  fair  contrast.  Bacteria,  as  well  as  animal  parasites, 
are  well  stained,  and  it  is  one  of  the  best  stains  for  the 
demonstration  of  parasites  in  tissues. 

(4)  Toluidin  Blue  is  a  stain  which  has  some  points 
of  resemblance  to  thionin.  The  stain  is  best  kept  as  a 
saturated  alcoholic  solution,  and  diluted  for  use  with 
twenty  parts  of  1  in  80  aqueous  solution  of  carbolic  acid. 
The  fixed  film  should  be  covered  with  the  stain  and  left 
for  ten  minutes  or  more.  It  is  difficult  to  over-stain,  and 
good  results  are  obtained  even  if  the  film  be  left  in  the 
stain  for  twenty-four  hours.  This  is  an  advantage,  as  the 
specimens  can  be  left  to  stain  whilst  other  occupations 
are  pursued.  The  main  advantage  of  the  stain  is  that 
the  pigment  is  less  obscured  than  in  specimens  stained 
by  carbol  thionin. 

If  blood  examinations  are  frequently  required,  it  is  well 
to  keep  the  stains  in  a  wide-necked  stoppered  bottle,  and 


74 


LEISHMAN'S   STAIN 


simply  place  the  slide  in  the  stain  for  the  time  required 
instead  of  putting  the  stain  on  the  slide.  Many  of  the 
stains  form  films  on  the  surface,  and  if  this  film  of  stain 
comes  into  contact  with  the  blood-film  it  will  adhere  to 
it.  The  bottle  of  stain  should  be  shaken  vigorously 
before  use.  The  fixing  agent  can  be  kept  in  the  same 
wav  (fig.  26). 


Fig.  26. 


Giemsa's  stain  also  gives  good  results  with  films  fixed 
in  absolute  alcohol,  but  better  results  are  obtained  if 
methylic  alcohol  is  used  for  fixation. 

B. — Staining  solution  also  fixes.  These  methods 
include  the  use  of  Louis  Jenner's  stain  and  the  stain 
used  in  Leishman's  modification  of  the  Romanowsky 
method,  as  in  both  of  these  the  methyl  alcohol  fixes 
the  film.  The  method  of  using  these  stains  for  the 
examination  of  normal  blood  has  been  already  described. 
It  suffices  for  the  demonstration  of  all  protozoa.  The 
stains,  particularly  Leishman's,  give  most  brilliant  results, 
and  show  more  points  in  the  structure  of  parasites  than 


DEH.-EMOGLOBINISED    FILMS  75 

any  other  method.  The  disadvantages  are  :  (1)  The 
necessity  of  having  distilled  water,  though  where  the 
rainfall  is  heavy  and  away  from  the  sea  rain-water  can 
often  be  used  ;  (2)  methyl  alcohol  is  very  volatile  ;  (3) 
the  stains,  under  circumstances  not  thoroughly  under- 
stood, seem  to  lose  their  strength  in  the  Tropics,  and 
consequently  are  not  so  universally  reliable  as  the  simpler 
stains  first  described. 

Louis  Jenner's  stain  in  particular  is  unreliable,  and 
seems  to  deteriorate  either  when  kept  in  the  solid  con- 
dition or  when  dissolved.  The  usual  failing  is  in  the 
basic  portion  of  the  stain,  and  unless  the  nuclei  of  the 
leucocytes  are  stained  a  brilliant  blue  the  stain  is  worth- 
less for  the  demonstration  of  parasites.  Leishman's  stain 
also  deteriorates,  but  can  be  made  satisfactorily  in  the 
manner  described.  Both  these  stains  must  be  rejected, 
however,  if  the  normal  constituents  of  the  blood  are  not 
satisfactorily  stained  by  them. 

The  results  obtained  by  the  use  of  these  stains  are  so 
clear  and  good  that  it  is  a  pity  to  discard  them,  and  a 
film  can  be  much  more  rapidly  examined  when  stained 
by  Leishman's  method  than  when  stained  by  any  other. 
The  worker  must,  however,  be  prepared  to  make  up  his 
own  stain,  and,  if  need  be,  to  distil  water  before  he  is 
justified  in  trusting  to  these  stains  alone. 

C. — No  preliminary  fixation.  When  parasites  are 
scanty  they  may  be  easily  overlooked  if  thin  films  only 
are  examined.  Thick  films,  if  fixed,  are  too  opaque  for 
examination  after  staining. 

A  useful  method  with  the  larger  parasites,  is  to  make 
a  very  thick  film  and  allow  it  to  dry.  When  dry  place 
in  water,  the  haemoglobin  will  be  dissolved  out  and 
only  parasites,  leucocytes,  blood  platelets  and  fibrin, 
with  the  decolorized  remnants  of  the  blood  corpuscles, 
will  be  left.  Such  decolorized  films,  when  dry,  can  be 
stained  with  any  of  the  basic  stains.  There  is  usually 
considerable  distortion  of  the  parasites,  but  many  of 
them,     particularly     crescents,     are     quite     recognizable. 


76  DEVELOPMENT   OF    H/EMOSPORIDIA 

Trypanosomas  can  be  more  readily  found  by  this  method 
than  in  thin  films,  but  are  so  much  distorted  that  results 
are  often  unreliable.  A  good  stain  to  use  with  these 
decolorized  films  is  carbol  fuchsin,  diluted  with  two 
parts  of  water.  Ross  prefers  to  decolorize  the  film  with 
a  weak  aqueous  solution  of  eOsin,  and  counter-stain 
with  a  weak  solution  of  polychrome  blue.  It  is  a  useful 
diagnostic  method,  but  not  suitable  for  obtaining  good 
specimens  of  the  more  delicate  parasites,  and  without 
considerable  practice  mistakes  are  frequently  made. 

General  Summary  of  the  Development  of  the 
H^emosporidia. 

The  Haemosporidia  are  parasitic  in  their  entire  exist- 
ence, and  require  for  complete  development  two  hosts : 
the  one  a  warm-blooded  animal,  and  the  other  usually  an 
insect.  In  the  warm-blooded  host  reproduction  takes 
place  asexually,  by  the  breaking  up  of  each  organism  into 
a  number  of  young  forms  or  spores. 

Each  of  these  spores  enters  a  red  corpuscle,  and  when 
it  has  reached  its  full  development  it,  in  turn,  breaks  up 
into  spores.  This  is  the  endogenous  or  asexual  cycle 
of  development — Schizogony.  The  host,  during  this 
cycle,  is  the  intermediate  HOST.  The  parasites  which 
develop  in  this  manner  are  known  as  SCHIZONTS,  and  the 
individual  spores  as  Merozoites. 

Some  of  the  merozoites,  however,  instead  of  becoming 
schizonts,  develop  into  the  sexual  or  GAMETOCYTE  form. 
These  do  not  reproduce,  or  undergo  any  further  change, 
whilst  in  the  intermediate  host.  If  they  are  taken  up  by 
the  definitive  host  they  become  sexually  active,  conjuga- 
tion takes  place,  and  further  development  follows.  The 
product  of  the  conjugation,  the  fertilized  female  or  zygote, 
increases  in  size  and  forms  a  cyst.  The  contents  of  this 
cyst  divide  into  several  masses,  SPOROBLASTS,  from  which 
small,  thread-like  bodies,  SPOROZOITES,  are  formed.  These 
bodies,  when  introduced  into  a  suitable  animal — the 
intermediate  host — become  schizonts. 


PARASITES   OF   MALARIA 


77 


This  cycle  is  a  sexual  one  and  is  known  as  SPOROGONY, 
and  the  host  during  this  period  is  therefore  the  defini- 
tive host.  Mosquitoes  belonging  to  several  genera  of 
the  Anophelince  are  therefore  the  definitive  hosts  of  the 
parasites  of  malaria,  whilst  man  is  the  only  known 
intermediate  host. 


Phase 
venous 


Fig.   27. 


The  diagram  (fig.  27)  represents  in  a  graphic  form  the 
two  methods  of  reproduction.  The  smaller  circle  repre- 
sents the  asexual  cycle  of  reproduction,  and  the  larger  the 
conjugation  of  the  male  and  female  sexual  forms,  with 
the  further  development  of  the  fertilized  female. 

Parasites  of  Malaria. — As  seen  in  fresh  living  blood 
the  youngest  form  of  malaria  parasite  is  a  small,  white, 
rounded  body  in  or  on  a  red  corpuscle.  A  clearer  por- 
tion can  sometimes  be  made  out  inside  it.  At  this  stage 
it  varies,  according  to  the  species  of  the  parasite,  from 


78  MALARIA    PARASI  I  ES 

one-eighth  to  one-quarter  the  diameter  of  the  red  blood 
corpuscle.  Even  the  youngest  forms  of  the  parasite 
often  show  at  the  edge  some  sign  of  amoeboid  movement. 
The  parasite  increases  in  si/.e,  and  the  amoeboid  move- 
ments become  pronounced.  A  parasite  seen  a  few  hours 
later  will  be  observed  to  be  not  only  larger,  but  to  have 
a  few  grains  of  pigment  scattered  about  inside  it.  The 
colour  of  the  pigment  and  the  size  of  the  grains  varies 
with  the  species  of  the  parasite.  When  the  parasite  has 
reached  the  fullest  growth  its  pigment  commences  to 
aggregate,  usually  towards  the  centre,  and  traces  of 
division  appear  in  the  surrounding  protoplasm.  These 
traces  of  division  become  obvious,  and  soon  the  central 
aggregation  of  pigment  can  be  seen  to  be  surrounded  by 
separate  rounded  masses  of  protoplasm — the  spores  or 
merozoites.  The  remnant  of  the  red  corpuscle  then  gives 
way  and  the  spores  are  poured  out  into  the  plasma.  A 
mature  parasite,  if  kept  under  observation  on  a  warm 
stage,  can  frequently  be  seen  to  break  up  in  this  manner. 

In  many  cases  a  leucocyte  will  appear  in  the  held 
and  devour  the  pigment,  and  often  some  of  the  spores. 
That  a  large  number  of  spores  are  destroyed  is  shown 
by  the  fact  that  though  each  tertian  parasite  forms 
eighteen  or  more  spores,  the  number  of  parasites  in  each 
successive  cycle  does  not  as  a  rule  increase. 

Free  spores  are  rarely  found  in  the  circulating  blood. 
Apparently  they  either  rapidly  take  shelter  in  a  red 
blood  corpuscle,  or  are  destroyed  by  leucocytes  or  in 
some  other  manner. 

Stained  specimens  demonstrate  some  further  points  in 
the  structure  of  the  parasites.  The  voungest  form,  the 
amoebula,  is  shown  to  consist  of  a  ring  of  protoplasm 
staining  with  basic  stains,  a  clear  unstained  space,  the 
vesicular  nucleus,  and  a  deeply  stained  spot,  or  nucleolus, 
usually  in  contact  with  the  ring  of  protoplasm. 

This  is  the  type  of  the  young  form  of  all  the  hsemo- 
sporidia.  They  are  all  composed  of  a  nucleolus  staining 
deeply,    a    "  vesicular "    nucleus,    which    does    not    stain 


MALARIA   PARASITES  79 

with  either  acid  or  basic  stains,  and  a  surrounding  proto- 
plasm which  stains,  but  much  less  intensely  than  the 
nucleolus,  with  basic  stains.  It  is  only  this  surrounding 
protoplasm  which  is  amoeboid,  and  consequently  in  the 
very  young  forms  the  range  of  amoeboid  movement  is  not 
very  great,  as  this  protoplasm  is  so  scanty. 

The  increased  growth  of  the  parasite  is  mainly  due 
to  the  increased  growth  of  the  ring  of  protoplasm,  though 
the  vesicular  nucleus  also  enlarges.  Pigment,  the  residue 
of  the  digested  haemoglobin,  is  deposited  in  this  proto- 
plasm only.  With  further  growth  the  vesicular  nucleus 
breaks  up  and  disappears,  and  all  that  is  seen  is  an 
irregularly  stained  parasite  with  pigment  scattered 
through  it.  Later  the  pigment  becomes  pushed  into 
one  block,  and  the  surrounding  protoplasm  is  seen  to 
be  divided  into  masses,  each  with  a  deeply  stained  spot, 
the  nucleolus  of  the  young  spore. 

Beyond  showing  the  "  ring  form,"  none  of  the  simple 
stains,  such  as  hematoxylin,  thionin,  methyl  blue,  &c, 
disclose  any  structural  changes  beyond  those  seen  in 
fresh  blood. 

Romanowsky's  method,  or,  better,  Leishman's  modi- 
fication of  this  method,  shows  more  markedly  the  struc- 
tural changes,  and,  in  particular,  the  varying  arrangement 
of  the  chromatin.  With  this  stain  the  youngest  form, 
amcebula  or  ring  form,  is  shown  to  have  the  chromatin 
arranged  as  a  solid  block  —  the  nucleolus — which  is 
stained  deep  ruby  red.  The  ring  of  protoplasm  stains 
blue,  whilst  the  vesicular  nucleus  is  unstained.  At  a 
later  stage  the  chromatin,  instead  of  being  in  a  solid 
mass,  is  seen  to  be  composed  of  scattered  points,  arranged 
at  part  of  the  periphery  of  the  vesicular  nucleus.  Still 
later,  when  the  vesicular  nucleus  disappears,  points  of 
chromatin  are  found  diffused  through  the  protoplasm. 
This  is  called  "  fragmentation  "  of  the  nucleolus.  Still 
later  the  chromatin  aggregates  into  small  masses  towards 
the  periphery,  and  a  secondary  division  of  the  masses 
takes  place,  resulting  in   the  formation   of  a   number  of 


8o  SPECIES   OF   MALARIA    PARASITES 

small  chromatin  nodules,  the  nucleoli  of  the  young 
spores. 

When  speculation  is  complete  each  of  these  chromatin 
nodules  is  situated  in  the  interior  of  a  portion  of  the 
protoplasm  of  the  parasite,  and  so  forms  the  spore  or 
merozoite.  The  pigment  takes  no  part  in  the  process,  and 
with  a  small  residual  portion  of  the  protoplasm  of  the 
parasite  is  pushed  into  a  mass,  usually  towards  the  centre 
of  the  group  of  spores.  When  the  corpuscle  bursts  and 
the  spores  are  liberated,  the  pigment  is  devoured  by 
leucocytes,  usually  the  large  mononuclear  leucocytes. 
These  thus  become  "  pigmented  leucocytes." 

The  chromatin  in  the  parasites  destined  to  become 
gametocytes  undergoes  different  changes.  The  first  stage 
is  the  same  as  in  the  young  parasites,  which  will  ultimately 
divide  asexual ly  into  spores — the  schizont.  The  chro- 
matin in  the  young  or  "ring"  form  of  the  gametocyte, 
as  of  the  schizont,  is  arranged  in  a  solid  block.  This 
chromatin  subsequently  divides  into  separate  granules, 
but  does  not  become  diffused  throughout  the  protoplasm 
as  it  does  in  the  schizont.  In  the  full-grown  gametocyte 
the  chromatin,  composed  of  numerous  particles  packed 
together,  forms  one  mass  in  the  interior  of  the  parasite, 
surrounded  by  a  zone  free  from  pigment  and  staining 
feebly.  The  changes  in  the  arrangement  of  the  chro- 
matin after  the  blood  is  shed  and  the  gametocytes  become 
sexually  active,  will  be  considered  with  the  sexual  or 
mosquito  phase  of  the  existence  of  the  malaria  parasite. 

All  the  human  malaria  parasites,  the  similar  parasites 
in  other  mammalia  and  birds,  as  far  as  is  known,  conform 
to  this  general  type. 

The  distinctive  points  on  which  the  division  o\  the 
human  parasites  into  distinct  species  is  made  are  as 
follows  : — 

(i)  Duration  of  the  asexual  cycle. 

(2)  Number  of  spores  formed  at  each  sporulation. 

(3)  Activity  of  movement. 

(4)  Preferential  sites  for  sporulation. 


MALARIA  Hi 

(5)  Differences  in  digestive  processes  in  different  para- 
sites as  indicated  by  the  differences  in  pigment. 

(6)  Effect  of  the  parasite  on  the  corpuscle  which 
contains  it. 

(7)  Shape  and  appearance  of  the  gametocyte. 

The  methods  of  examination  described  are  ample  for 
determining  these  points. 

(1)  The  length  of  cycle  can  be  readily  ascertained  in 
the  cast;  of  parasites  which  sporulate  in  the  peripheral 
blood.  The  blood  is  examined  at  intervals,  so  as  to 
determine  the  length  of  time  between  the  sporulation 
of  a  group  of  the  parasites  and  the  steady  growth  of 
this  group  up  to  the  next  period  of  sporulation.  In 
benign  tertian  and  quartan  this  is  readily  done,  and  it 
will  be  found  that  the  period  or  length  of  cycle  is 
approximately  forty-eight  and  seventy-two  hours  respec- 
tively. It  is  difficult  to  determine  in  malignant  tertian 
(aestivo-autumnal  or  sub-tertian)  malaria,  as  only  the 
young  schizonts  and  mature  gametocytes  are  common 
in  the  peripheral  blood.  The  period  for  this  species  is 
certainly  variable,  and  the  parasites  are  commonly  in 
several  stages  of  growth,  so  that  periodicity  is  not  so 
clearly  defined  as  in  the  other  species  of  parasites  of 
human  malaria. 

(2)  The  number  of  spores  can  be  counted  in  the  fresh 
or  stained  blood  when  the  parasites  are  fully  mature. 
If  stained  for  chromatin,  the  number  of  spores  can  be 
counted  earlier.  It  will  be  found  that  in  benign  tertian 
the  spores  are  usually  about  20,  but  may  be  as  low  as 
15  or  as  high  as  25,  or  even  more.  In  benign  quartan 
12  is  a  maximum  rarely  exceeded,  whilst  8,  9,  or  10  are 
the  common  numbers.  The  number  in  sub-tertian  is 
more  variable — 7  to  30. 

(3)  The  activity  of  the  amoeboid  movement  can  only 
be  determined  with  certainty  in  the  living  blood.  In- 
ternal movement  in  the  parasite  itself  is  also  shown  in 
the  fresh  fluid  blood  by  movement  of  the  pigment  in 
the  parasite. 

6 


82  .MALARIA 

Amoeboid  movements  can  be  inferred  in  stained 
specimens,  as  the  parasites  present  great  varieties  in 
shape,  and  frequently  where  amoeboid  movements  have 
been  active  when  the  film  is  dried,  the  pseudopodia  can 
still  be  seen. 

(4)  The  selective  site  for  sporulation  is  of  great  impor- 
tance, as  one  species,  the  malignant  tertian  (sub-tertian) 
sporulates  almost  exclusively  in  the  internal  organs,  and 
the  occasional  malignant  clinical  course  of  the  disease 
caused  by  this  parasite  is  due  to  the  selection  of  the 
brain  or  other  important  organ  as  a  site  for  sporulation. 

The  absence  of  full-grown  forms  and  the  determination 
of  the  absence  of  sporulating  forms  indicate  that  the 
parasites  are  sporulating  elsewhere,  i.e.,  in  the  internal 
organs.  Post-mortem  examination  of  fatal  cases  shows 
in  which  organs  the  sporulating  parasites  are,  but  the 
clinical  symptoms  often  give  a  clue. 

Benign  tertian  parasites  sporulate  to  a  considerable 
extent  in  the  circulating  blood,  though  the  splenic  sinuses 
are  their  preferential  resort  at  this  period.  Quartan 
parasites  sporulate  freely  in  the  circulating  blood,  whilst 
sub-tertian  (malignant  tertian)  is  hardly  ever  found  sporu- 
lating except  in  the  visceral  capillaries. 

All  the  phases  of  benign  tertian  and  quartan  can  be 
observed  in  the  blood  obtained  by  pricking  the  finger  or 
ear,  and  therefore  the  determination  of  the  length  of  the 
cycle  with  these  parasites  is  easy.  With  malignant  tertian, 
on  the  other  hand,  the  stages  of  sporulation,  or  even 
the  full-grown  schizonts,  are  rarely  to  be  observed  in 
the  peripheral  blood.  The  full-grown  gametocytes  are 
common  in  the  blood,  but  the  intermediate  stages  of 
growth  cannot  be  found  except  in  the  visceral  capillaries. 
Puncture  of  the  spleen  in  the  living  subject  may  show 
these  forms.  If  undertaken  aseptically  the  operation  is 
considered  to  be  practically  free  from  risk  to  the  patient ; 
but  as  accidents  have  occurred  this  method  should  not 
be  employed  except  in  cases  where  certainty  of  diagnosis 
is  absolutely  necessary. 


MALARIA  83 

In  fatal  cases  with  cerebral  symptoms,  the  sporulating 
and  full-grown  forms  can  be  observed  in  enormous 
numbers  in  the  brain  and  often  in  other  organs — lungs, 
suprarenals,  liver,  &c.  In  other  fatal  cases  they  may 
be  found  in  greatest  numbers  in  the  intestinal  mucosa, 
pancreas,  and  rarely  in  the  kidneys. 

The  organ  in  which  the  parasites  are  most  commonly 
found  post  mortem  is  the  brain,  and  cerebral  symptoms 
are  common  in  so  many  cases  that  recover  that  it  seems 
probable  that  this  is  a  favourite  site.  It  must  be  remem- 
bered, however,  that,  as  the  blocking  of  the  cerebral 
capillaries  is  the  most  common  cause  of  death  in  acute 
malaria,  the  proportion  of  fatal  cases  with  this  com- 
plication gives  an  exaggerated  idea  of  the  frequency  with 
which  this  site  is  selected  by  the  parasites. 

For  diagnostic  purposes  it  suffices  to  take  a  small  por- 
tion of  the  fresh  brain  substance  and  squash  it  between 
the  slide  and  cover-glass.  The  capillaries  in  a  case  of 
cerebral  malaria  will  then  be  seen  to  be  filled  with  grains 
of  black  pigment.  Though  the  parasites  themselves 
cannot  be  seen,  these  grains  of  pigment  are  diagnostic* 
as  they  are  contained  in  the  full-grown  or  sporulating 
parasite. 

It  is  not  absolutely  necessary  to  open  the  skull,  though 
it  is  better  to  do  so.  The  needle  of  a  large  exploring 
syringe  can  be  forced  through  the  orbital  plate  of  the 
frontal  bone  and  the  brain  stirred  up  a  little ;  suction 
with  the  syringe  will  then  usually  bring  away  sufficient 
brain  matter  for  examination.  As  the  puncture  is  made 
through  the  conjunctiva  no  disfigurement  results,  and 
the  site  of  puncture  will  be  covered  by  the  eyelid. 

The  vessels  on  the  pin  mater,  particularly  at  the  base  of 
the  brain,  are  frequently  pigmented.  This  pigmentation 
must  not  be  confused  with  malarial  pigmentation.  The 
pigment  is  not  contained,  as  it  is  in  malaria,  in  the  capil- 
laries, but  in  their  walls,  and  is  insoluble  in  alkalies  which 
readily  dissolve  melanin.  The  finely  granular  or  streaky 
arrangement   of   this    natural   pigmentation    differs  from 


84  MALARIA 

the  coarser  arrangements  of  the  melanin  particles,  and 
the  colour  is  brown,  not  black.  This  pigmentation, 
non-malarial,  occurs  in  all  races,  but  is  commoner  in  the 
coloured  races.  It  is  found  in  new-  or  still-born  children 
whose  organs  are  free  from  malarial  pigmentation. 

To  demonstrate  the  arrangement  of  the  pigment 
granules  of  malaria  in  a  hardened  brain,  thick  sections 
should  be  cut.  These  can  be  quite  easily  cut  by  hand, 
and  without  any  staining  passed  through  absolute  alcohol 
and  then  oil  of  cloves  to  dissolve  the  fatty  brain 
constituents  and  render  the  section  transparent.  The 
section  can  then  be  mounted  in  balsam,  and  in  a  malarial 
case  every  capillary  will  then  be  seen  to  be  mapped  out 
by  the  contained  pigment  granules  almost  as  if  it  had 
been  injected. 

These  methods,  though  useful  for  rapid  diagnosis,  do 
not  show  the  parasite.  With  the  fresh  brain  specimens, 
whether  a  squashed  fragment  or  a  fragment  drawn  out 
with  the  exploring  springe  be  examined,  parasites  will 
often  be  seen  in  corpuscles  which  have  escaped  from  the 
capillaries. 

To  show  the  parasites  well  it  is  necessary  to  stain 
them.  With  the  fresh  brain  it  is  not  necessary  to  cut 
sections  nor  is  it  advisable.  A  smear  should  be  made 
of  the  brain  substance,  and  this  should  be  dried  rapidly 
by  waving  it  in  the  air — not  by  the  application  of  heat. 
The  smear  need  not  be  very  thin,  as  the  greater  part  of 
the  brain  matter  is  subsequently  dissolved.  The  smear 
can  be  stained  by  Leishman's  method,  but  must  then 
be  thoroughly  dried  to  dehydrate,  and  mounted  in 
xylol  balsam.  This  method  shows  the  chromatin  in  the 
parasites,  but  the  drying  causes  much  distortion  of  the 
surrounding  tissues. 

If   this    method    be    not    adopted,    hematoxylin   gives 

good  and  permanent  results,  and  carbol  thionin  also  gives 

"  very   good    results.     The   procedure   is    as  follows  :  Fix 

the  smear  in  absolute  alcohol  or  alcohol  and  ether  for  ten 

minutes  and  allow  to  dry. 


MALARIA  85 

To  stain  with  hematoxylin,  cover  the  smear  with  a 
hae  matin  solution  and  leave  for  ten  minutes.  Flush 
off  the  stain  and  place  the  slide  in  water  for  five  minutes. 
Dehydrate  with  spirit  and  oil  of  cloves.  Mount  in  xylol 
balsam. 

With  carbol  thionin  the  procedure  is  rather  more 
complicated  and  requires  more  care.  It  is,  however, 
a  general  method,  and  is  a  suitable  one  also  for  the 
demonstration  of  vegetable  micro-organisms  in  tissues. 

Fix  the  smear  in  absolute  alcohol  as  before,  and  cover 
it  with  the  strong  carbol  thionin  solution.  Leave  for 
ten  to  fifteen  minutes.  It  is  essential  that  at  this  stage 
the  specimen  should  be  very  much  over-stained,  as  much 
stain  is  lost  in  the  subsequent  processes.  Flush  off  the 
stain  with  water.  Pass  rapidly  through  methylated 
spirit,  not  absolute  alcohol.  Much  stain  will  come 
awTay,  and  care  must  be  exercised  that  the  specimen 
is  still  over-stained  when  removed  from  the  spirit.  The 
time  the  specimen  is  left  in  the  spirit  is  determined 
entirely  by  the  colour.  It  cannot  be  completely  dehy- 
drated at  this  stage,  or  too  much  colour  would  be 
removed.  Drain  off  and  gently  blot  off  excess  of  spirit. 
Cover  with  oil  of  cloves  and  place  under  the  micro- 
scope. The  oil  of  cloves  will  dissolve  out  the  brain 
fatty  matter,  complete  the  dehydration,  and  slowly 
remove  the  excess  of  stain.  When  it  is  observed  that 
nearly  enough  stain  is  removed,  a  cover-glass  can  be 
placed  over  the  specimen  and  an  examination  made 
with  an  oil  immersion  lens.  If  the  specimen  is  well 
stained  the  cover-glass  can  be  removed,  and  the  speci- 
men placed  in  xylol  to  remove  the  oil  of  cloves,  which 
would  otherwise  ultimately  decolorize  the  specimen. 
Finally  it  is  mounted  in  xylol  Canada  balsam. 

Specimens  of  brain  hardened  in  absolute  alcohol  can 
be  used.  Thin  sections  are  required,  embedded  in 
paraffin  for  choice.  The  processes  are  the  same  as  for 
brain  smears  after  the  paraffin  has  been  removed  from 
the   specimen   by   xylol,    the   xylol  by  alcohol,  and   the 


86  MALARIA-SECTIONS 

alcohol  by  water ;  but  the  section  must  not  be  allowed 
to  dry  at  any  stage. 

Van  Gieson's  method  is  also  useful  for  staining  parasites 
in  tissues.  Van  Gieson's  solution  is  composed  of  i^  per 
cent,  acid  fuchsin  dissolved  in  a  saturated  aqueous  solu- 
tion of  picric  acid. 

The  method  is  as  follows  :  Remove  the  paraffin  with 
xylol,  the  xylol  with  spirit,  and  the  spirit  with  water  in 
the  usual  way.  Stain  with  hematoxylin  or  haemalum  for 
ten  to  fifteen  minutes  and  flush  off  the  stain  with  tap 
water.  Leave  the  specimen  in  tap  water  for  five  minutes 
to  "blue."  Then  treat  with  van  Gieson's  solution  for 
half  to  one  minute,  not  longer.  Wash  this  solution  off 
with  spirit,  not  with  water,  clear  with  oil  of  cloves,  and 
after  removing  the  oil  of  cloves  with  xylol,  mount  in 
xylol  balsam. 

The  parasites  and  the  nuclei  of  cells  are  stained  with 
the  haematoxylin,  the  protoplasm  of  the  tissue  cells  with 
the  picric  acid,  whilst  fibrous  tissue  is  stained  red  with 
the  acid  fuchsin. 

The  parasites  in  sections  show  well,  but  are  smaller, 
only  about  half  the  size  of  those  in  the  smears  made 
from  the  fresh  brain,  as  the  fixative  agent  causes  much 
shrinking  (Plate  IV.,  yi,  \a).  As  this  parasite  is  the 
smallest  of  the  human  malaria  parasites,  and  when  full- 
grown  often  little  more  than  half  the  diameter  of  the 
red  blood  corpuscles,  there  is  in  these  shrunken  speci- 
mens considerable  difficulty  in  making  out  the  spores  into 
which  the  parasites  are  broken  up. 

In  these  specimens  the  corpuscle  containing  the  para- 
site is  not  lying  singly  or  flat,  as  it  is  in  the  blood-film, 
or  smear,  but  is  one  of  the  many  corpuscles  packed  into 
the  capillary,  so  that  it  is  exceptional  for  the  outline 
of  the  corpuscle  containing  the  parasite  to  be  made  out 
(fig.  28). 

These  methods  are  general  methods  for  the  demon- 
stration of  protozoa  in  tissues.  With  carbol  thionin 
bacteria  are  also  shown. 


<F*  «&<§ 


^  /ft — r£ 


*   *••• 


V   %#l 


5;-, 


•@<. 


*     *1      f  - 


? 


--":' 


fr        : 


a. — Pancreas, 


*     —  * 

£. — From  Intestine. 

Fig.  28. 

*/.  86. 

MALARIA-SECTIONS  S1/ 

In  the  large  vessels  parasites  are  not  so  common.  In 
the  small  vessels  the  corpuscles  containing  the  parasites 
are  often  found  only  in  contact  with  the  wall  of  the 
vessel,  and  no  parasites  are  contained  in  the  corpuscles 
towards  the  centre  of  the  vessel.  The  largest  number 
of  corpuscles  containing  parasites  are  in  the  capillaries. 

This  occurrence  in  the  minute  capillaries  results  in 
a  blood  stasis  more  or  less  complete.  Such  a  stasis 
involving  a  large  part  of  the  brain  results  in  headache, 
drowsiness,  and  coma  in  adults,  rarely  delirium,  and 
in  convulsions  and  coma  in  young  children,  and  is  the 
most  common  cause  of  death  in  acute  malaria.  The 
process  is  often  spoken  of  as  thrombosis.  This  is  in- 
correct ;  there  is  no  coagulum  formed  ;  no  fibrin,  and 
the  leucocytes  are  not  aggregated  in  the  capillaries  and 
take  no  part  in  the  process.  Clinically,  where  active 
treatment  is  adopted  we  have  abundant  evidence  that 
the  condition  is  a  transient  one.  Speedy  and  complete 
recovery  from  the  condition  of  complete  coma  frequently 
takes  place  under  energetic  treatment  with  quinine. 

The  parasites  themselves  are  usually  at  different  stages. 
Quite  young  parasites,  hardly  larger  than  the  spores,  may 
be  found.  More  commonly  the  great  majority  of  the 
parasites  contain  centralized  pigment  and  have  lost  their 
vesicular  nucleus.  In  some  specimens  a  large  proportion, 
in  others  a  small  proportion,  will  be  found  sporulating. 

The  number  of  spores  varies  greatly,  and  in  some 
specimens  only  seven  or  eight  spores  will  be  found  to 
each  parasite.  In  other  cases  the  number  will  be  twenty 
or  more. 

This  variation  in  the  number  of  spores  is  one  of  the 
distinctions  on  which  reliance  has  been  placed  for  the 
subdivision  of  this  species  into  three. 

In  most  parts  of  the  body  a  temporary  partial  stasis 
of  the  blood  in  the  capillaries  leads  to  no  sudden  fatal 
changes  or  symptoms,  and  consequently,  unless  the  brain 
is  also  involved,  a  fatal  result  is  not  common.  There 
are,    however,   peculiar  risks  attending  another  region — 


88  MALARIA 

the  intestines.  Stasis  occurring  in  the  capillaries  of  the 
mucosa  impairs  the  vitality  of  the  cells  and  renders  them 
liable  to  be  invaded  by  some  of  the  bacterial  contents 
of  the  alimentary  tract.  Secondary  inflammation  and 
superficial  necrosis  may  thus  result,  and  so  indirectly,  by 
lowering  the  nutrition  of  the  mucosa,  a  fatal  enteritis 
may  be  set  up.  There  is  some  reason  for  believing 
that  sufficient  attention  has  not  been  paid  to  the  indirect 
results  of  repeated  blood  stasis  in  the  various  viscera, 
consequent  on  malarial  infection. 

The  parasites  in  these  and  other  situations  are  best 
demonstrated  in  sections  stained  with  hematoxylin, 
carbol  thionin,  or  Van  Gieson,  as  already  described  for 
the  brain.  Carbol  thionin  has  the  additional  advantage 
of  staining  the  micro-organisms  which  have  invaded  the 
mucosa.  These,  however, 'can  be  shown  in  separate 
specimens  somewhat  better,  particularly  those  micro- 
organisms which  retain  their  stain  when  treated  by 
Gram's  method. 

(5)  Melanin  malarial  pigment,  or  simply  "pigment," 
is  the  residue  from  the  digestion  of  haemoglobin,  and 
contains  the  excess  of  iron  over  the  minute  amount 
required  by  the  parasite. 

In  the  different  species  it  is  deposited  in  different 
forms.  In  the  quartan  it  is  deposited  as  granules,  which 
are  coarse  and  black,  and  in  the  benign  tertian  the  colour 
varies  from  a  yellow-brown  to  a  dark  brown,  while  it  is 
always  in  fine  granules  ;  in  the  sub-tertian  the  pigment 
is  not  commonly  seen  in  the  early  forms  present  in  the 
peripheral  blood.  When  it  is  found  it  is  in  fine,  black 
granules,  which  aggregate  into  a  mass  earlier  than  in  the 
other  forms  of  parasites. 

(6)  The  parasite  affects  the  corpuscle  containing  it  in 
different  ways. 

In  quartan  fever,  although  the  parasite  is  in  the 
interior  of  the  corpuscle,  the  bulk,  or  at  any  rate  the 
diameter  of  a  corpuscle  containing  the  parasite,  is  slightly 
below  the  average    in    the    majority    of    instances.     The 


MALARIA 


89 


colour  of  the  red  corpuscle  is  not  lighter,  and  is  fre- 
quently a  trifle  darker  than  the  average  of  the  red 
corpuscles. 

In  benign  tertian  there  is  a  great  difference,  as  the 
diameter  of  the  corpuscle  is  decidedly  above  the  average 
and  the  corpuscle  is  pale.  This  is  well  seen  both  in 
stained  and  unstained  specimens.  The  corpuscle  is  easily 
distorted,  and  is  consequently  frequently  compressed  by 


Fig.  29. — a  to/,  Phases  in  the  asexual  development  of  the  quartan 
parasite  ;  x  to  z,  phases  in  the  sexual  development. 


neighbouring  blood-cells,  when  these  are  in  contact  with 
it.  Leishman's  and  some  other  similar  stains  reveal  still 
further  changes,  as  granules  staining  deeply  red  are 
found  throughout  the  corpuscle  in  parts  not  invaded  by 
the  parasites.  These  granules  are  small  and  not  very 
distinct  when  the  parasite  is  very  young,  but  with  the 
growth  of  the  parasite  become  more  numerous  and  more 
distinct  (Plate  VI.,  7,  8,  9).      They  are  not  found  with 


9o 


MALARIA 


the  other  human  malaria  parasites,  nor  with  the  haema- 
mcebidae  of  mammals  and  birds,  but  are  found  in 
corpuscles  infected  by  one  species  of  hasmogregarine 
in  the  frog  (Plate  V.,  21.) 

To  show  them  well,  the  mixed  water  and  stain  in 
Leishman's  method  must  be  left  on  double  the  normal 
time,  and  fifteen  minutes  is  not  too  long  with  most 
specimens  of  the  stain. 


Fig.  30. — a  to/,  Phases  in  the  asexual  development  of  the  benign  tertian 
parasite  ;  itoz,  phases  in  the  sexual  development. 

These  granules  are  known  as  Schiiffner's  granules  or 
dots,  and  must  not  be  confused  with  the  basophilic 
granules,  which  stain  blue  by  the  same  method,  found  in 
some  instances  abundantly  in  malarial  blood,  as  well  as 
in  other  diseases. 

The  effect  of  the  malignant  tertian  parasite  on  the 
corpuscle  is  variable.  The  young  forms  met  with  in 
peripheral  blood  do  not  appreciably  affect  it,  but  occa- 
sionally the  corpuscle  is  changed  in  colour  to  a  yellowish 


MALARIA  91 

or  brassy  tinge.  With  the  more  advanced  stages  of  the 
parasite  the  corpuscle,  without  increasing  in  size,  becomes 
more  or  less  decolorized.  In  the  full-grown  parasite, 
whether  schizont  or  gametocyte,  the  remnant  of  the  red 
corpuscle  is  a  mere  colourless  shadow,  which  is  not 
always  easy  to  define  (Plate  IV.,  10  to  19). 

(7)  The  shape  of  the  gametocyte  in  benign  tertian  and 
quartan  is  that  of  a  rounded  body  indistinguishable  by 
its  shape  from  the  full-grown  schizont  before  sporula- 
tion  has  taken  place.  It  can  be  distinguished  by  the 
presence  of  a  clear  space,  free  from  pigment  in  the  un- 
stained specimen.  In  malignant  tertian  the  gametocyte 
is  the  so-called  "  crescent."  It  is  better  described  as 
sausage-shaped.  It  is  not  truly  crescent-shaped,  as, 
though  often  slightly  curved,  the  two  ends  are  broad  and 
rounded,  not  tapering  to  sharp  points  as  in  a  true  cres- 
cent. These  bodies  always  contain  pigment,  and  this 
pigment  is  never  in  a  solid  block,  but  is  always  composed 
of  discrete  particles  grouped  in  a  cluster  near  the  centre 
of  the  parasite. 

These  gametocyte  forms  are  best  observed  in  fresh 
fluid  blood-films,  and  it  is  only  in  such  films  that  the 
subsequent  changes  can  be  followed. 

As  it  is  not  easy  to  distinguish  the  gametocyte  of 
benign  tertian  and  quartan  malaria  from  the  sporocyte, 
the  subsequent  changes  are  most  readily  observed  in  the 
case  of  malignant  or  sub-tertian  malaria,  as  the  peculiar 
"crescent"  shape  renders  the  identification  of  the 
gametocyte  easy  in  this  form  of  malaria. 

These  changes  only  take  place  when  alterations  in 
the  blood  occur,  such  as  abstraction  or  addition  of  water. 
The  crescents  lose  their  peculiar  shape,  throw  off  the 
remains  of  the  containing  corpuscles,  and  become  first 
oval  and  then  spherical.  Small  portions,  one  or  two, 
are  extruded,  the  polar  bodies,  and  remain  usually 
adherent  to  the  outer  surface  of  the  altered  crescent. 

Of  these  altered  crescents,  a  proportion  which  varies  in 
different  specimens  throw  out  long,  filamentous  flagella, 


92 


GAMETOCYTES 


varying  in  number  from  two  or  three  to  six.  These 
flagella  are  actively  motile  and  lash  about  in  the  blood 
plasma,  or  over  neighbouring  red  corpuscles  for    some 


Fig.  31. — a  to  e,  Phases  in  the  asexual  development  of  the  malignant 
malarial  parasite ;  x  to  z,  phases  in  the  sexual  development — x  y  z,  of  the 
female  :  xx  j/1  z1,  of  the  male. 

time.     Finally  they  break  away,  and  can  be  seen  moving 
rapidly  through  the  plasma. 

The  crescents  which  undergo  this  change  and  flagellate 
are  the  males,  and  the  flagella  are  equivalent  to  sperma- 
tozoa, and  are  known  as  MiCROGAMETES.  The  residue 
of  the  crescent  is  a  small  protoplasmic  mass  containing 


GAMETOCYTES  93 

all  the  pigment.     It  soon  dies,  and  is  either  broken  up  or 
devoured  by  a  leucocyte. 

The  other  altered  crescents  do  not  flagellate.  After  the 
extrusion  of  the  polar  bodies  they  retain  their  spherical 
shape,  but  the  pigment  in  the  interior  is  often  in  a  state 
of  violent  agitation.     They  form  the  MACROGAMETES. 

Very  rarely  a  flagellum  that  has  broken  off  the  male 
crescent  is  seen  to  enter  this  rounded  body,  and  is  ab- 
sorbed by  it.  These  mature  crescents  which  do  not 
flagellate  are  the  females,  and  the  entrance  of  the  flagellum 
is  a  process  of  fertilization.  After  fertilization  further 
changes  take  place,  the  pigment  is  thrown  into  violent 
agitation,  the  whole  body  changes  shape,  one  end  becomes 
conical,  and  the  body  becomes  actively  motile,  moving 
steadily  through  the  blood  serum. 

This  fertilized  female,  as  it  is  the  product  of  conjugation, 
is  a  zygote,  but  is  known,  whilst  motile,  as  the  "  travell- 
ing vermicide"  or  ookinet,  and  passes  into  the  outer 
wall  of  the  mosquito's  stomach  beneath  the  epithelial 
lining  where  it  becomes  encysted,  and  for  this  stage  the 
general  term  zygote  only  is  employed. 

The  male  and  female  crescents  can  often  be  distin- 
guished in  the  freshly-shed  blood  by  the  arrangement  of 
the  pigment.  In  the  female  there  is  usually  a  clear  space 
in  the  middle  surrounded  by  pigment,  whilst  in  the  male 
no  such  clear  space  is  present  and  the  pigment  is  less  in 
a  ring  and  more  in  a  clump  than  in  the  female. 

The  young  forms  of  crescents  are  sometimes  found  in 
the  brain,  spleen  and  elsewhere  ;  they  can  be  distin- 
guished by  their  shape  and  the  tendency  of  the  pigment 
to  be  arranged  in  a  central,  irregular  clump,  and  not  in 
one  mass. 

The  same  series  of  changes  occur  in  the  gametocytes  of 
benign  tertian  and  quartan  malaria  after  the  blood  is  shed. 

The  gametocytes  stain  rather  feebly  with  basic  stains. 
The  outlines  of  the  red  corpuscles  which  contain  them 
can  usually  be  made  out,  though,  as  nearly  all  the 
haemoglobin  is  absorbed,  the  remnant  does  not  stain,  or 
only  faintly,  with  eosin. 


94  GAMETOCYTES 

The  gametocytes  contain  chromatin  in  considerable 
quantity,  but  this  chromatin,  though  it  stains  with  the 
red  of  the  polychrome  methylene  blue,  does  not  stain 
with  hsematoxylin  or  most  basic  stains. 

Stained  by  Leishman's  method,  the  gametocytes  of 
benign  tertian  and  quartan  are  easily  recognized,  as  the 
chromatin  granules  are  collected  in  a  clump  surrounded 
by  an  unstained  area  free  from  pigment  (Plate  VI.,  6). 

In  the  crescents  the  chromatin  in  the  female  is  collected 
into  a  solid  block  in  the  centre,  and  round  this  the  pig- 
ment is  arranged.  In  the  male  there  is  no  central  block 
of  chromatin,  but  numerous  scattered  particles  are  mixed 
up  with  the  pigment.  In  the  males  the  chromatin  may 
be  very  abundant  (Plate  VI.,  18,  19). 

To  make  permanent  preparations  showing  the  changes 
that  occur  in  shed  blood,  it  is  necessary  to  prevent  the 
blood  from  drying  and  to  examine  at  intervals  of  a  few 
minutes.  For  this  purpose  the  slides  with  the  fluid  film 
on  them  must  be  kept  in  a  moist  chamber.  This  is 
easily  done  by  cutting  windows  in  a  folded  piece  of 
blotting  paper  and  placing  this  blotting  paper  on  a  slab. 
The  blotting  paper  should  be  moistened,  and  the  windows 
each  covered  with  a  slide  on  which  is  the  wet  film  face 
downwards.  The  evaporation  from  the  damp  blotting 
paper  will  render  the  air  so  moist  that  evaporation  from 
the  film  will  be  very  slow. 

One  of  these  slides  can  be  taken  off  and  allowed  to 
dry  every  five  minutes,  and  in  this  way  we  have  a  series 
of  blood-films  five,  ten,  fifteen,  twenty  minutes,  or  more, 
after  the  blood  has  been  shed. 

To  obtain  stained  specimens  of  flagellating  bodies  thick 
films  can  be  used,  and  when  allowed  to  dry  the  haemo- 
globin may  be  removed  by  placing  the  slide  in  water. 
After  this  the  film  is  again  allowed  to  dry,  fixed  in 
alcohol,  and  stained  with  a  strong  basic  stain,  such  as 
carbol  fuchsin. 

To  observe  well  the  changes  that  occur  in  the  arrange- 
ment  of    the  chromatin    these    decolorized    films  cannot 


GAMETOCYTES  95 

be  used,  but  the  specimen  with  a  moderately  thick  film 
must,  after  drying,  be  stained  by  Leishman's  method. 
In  both  male  and  female  a  portion  of  the  chromatin 
will  be  seen  to  be  extruded  in  the  polar  body.  The 
remainder  increases  in  amount,  and  most  of  it  in  the 
male  will  be  seen  in  the  form  of  nodules  at  the  periphery 
of  the  altered  crescent.  Finger-like  processes  of  the 
protoplasm  will  be  seen  to  project  from  the  vicinity  of 
these  masses,  at  first  without  any  chromatin,  and  these 
processes  elongate  and  form  long,  slender  flagella  without 
chromatin.  Ultimately,  however,  the  chromatin  enters 
the  flagella  as  a  long,  thin  filament,  leaving  a  mere 
residuum  in  the  remnant  of  the  crescent  at  the  base  of 
the  flagellum.  When  the  flagellum  breaks  loose  it  has 
this  chromatin  filament  running  nearly  its  whole  length. 
Even  when  all  the  flagella  have  broken  away  there  are 
still  remnants  of  the  chromatin  in  the  protoplasmic 
residual  mass  left  behind. 

In  the  female  the  chromatin  forms  a  less  compact 
mass  after  the  extrusion  of  the  polar  bodies,  and  it  is 
with  this  mass  that  the  chromatin  of  the  flagellum  which 
fertilizes  it  probably  fuses. 

Minor  differences  in  the  crescents  as  regards  shape, 
staining  reaction,  and  colour  of  the  pigment  are  described 
by  those  who  subdivide  the  malignant  or  sub-tertian 
into  two  or  more  species. 

As  regards  the  genesis  of  the  gametocytes,  suggestions 
have  been  made  from  time  to  time  that  they  may  be 
formed  by  the  union  of  two  young  parasites  in  one  cor- 
puscle. Two,  or  even  three  or  four,  parasites  are  not 
uncommonly  found  in  one  red  corpuscle.  These  para- 
sites may  be  in  actual  contact  with  each  other,  but 
there  is  no  satisfactory  evidence  that  conjugation  or 
fusion  of  two  such  parasites  ever  takes  place.  On  general 
grounds  there  is  little  or  nothing  to  support  this  hypo- 
thesis of  the  formation  of  the  male  and  female  sexual 
forms.  Schaudinn  believed  that  the  development  of  the 
gametocyte  was  slower  than  that  of   the  sporocyte.     As 


96  GAMETOCYTES 

occasionally  we  find  a  mature  sporocyte  and  a  full  grown 
gametocyte  in  the  same  cell  this  is  not  probable. 

The  differences  between  the  three  main  species  of 
parasites  are  shown  in  tabular  form  on  the  next  page 
(Table,  p.  97.) 

Mistakes  can  be  made  with  every  method  of  blood 
examination,  but  most  of  them  after  a  little  experience 
are  easily  avoided. 

In  fresh  fluid  blood-films  the  following  are  often  mis- 
taken for  young  non-pigmented  parasites. 

(1)  The  normal  lighter  colour  of  the  central  portion 
of  the  corpuscle,  due  to  the  bi-concave  shape  of  the  red 
corpuscle.  The  gradual  shading  and  the  absence  of  any 
definite  edge  to  the  lighter  part  is  usually  sufficient  to 
prevent  this  error,  and  familiarity  with  this  appearance 
in  normal  blood  is  of  importance. 

(2)  Vacuoles  or  slits  in  a  blood  corpuscle  are  distin- 
guished by  the  very  sharp,  abrupt  edge  of  such  a  vacuole 
and  by  the  oscillatory  motion  of  the  edge.  It  can  be 
generally  seen  that  whilst  in  a  parasite  there  is  a  faint 
opalescence,  in  the  vacuole  the  space  is  perfectly  clear 
{vide  fig  19,  c). 

(3)  Blood  plates  resting  on  a  blood  corpuscle  are  in 
some  cases  difficult  to  distinguish.  Round  such  blood 
plates  there  is  usually  a  ring  or  "  halo  "  where  the  haemo- 
globin has  been  pressed  away,  and  in  some  cases  by 
focussing  it  can  be  determined  that  the  body  is  one  which 
is  on  and  not  a  part  of  the  red  corpuscle. 

(4)  Small  particles  resting  on  a  corpuscle  will  displace 
the  haemoglobin  beneath  them  and  cause  a  lighter- 
coloured  patch  in  the  corpuscle.  Such  particles,  if 
dark,  are  often  mistaken  for  pigment,  and  the  pale  area 
is  taken  for  the  parasite. 

(5)  Crenations,  particularly  when  they  occur  as  pro- 
jections on  the  upper  or  lower  surface  of  a  corpuscle,  are 
frequent  sources  of  error.  The  effect  of  focussing,  or 
alteration  of  the  illumination,  will  show  the  true  nature 
of  these  crenations  (vide  fig.  19,  a,  b). 


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98  .  DECEPTIONS 

(6)  Bent  or  twisted  "  buckled"  corpuscles  may  cause 
confusion  (fig.  19,  d  and  e). 

Many  effects  are  mistaken  for  pigmented  parasites. 
Some  of  these  are  due  to  insufficient  illumination,  as 
refraction  effects  with  a  dim  light  closely  simulate  grains 
of  pigment.  Crenated  corpuscles,  leucocytes,  &c,  are 
thus  sometimes  taken  for  pigmented  parasites.  Full 
illumination  will  dispel  this  illusion.  Particles  of  dirt, 
or  epithelial  fragments  with  specks  of  dirt  adhering, 
usually  overlap  at  one  edge  or  other  the  red  corpuscle  on 
which  they  lie.  If  they  do  not,  by  focussing  it  can  often 
be  determined  that  they  lie  on  or  beneath  the  red  cor- 
puscle. In  most  cases  such  fragments  can  be  distin- 
guished by  their  sharp  angular  outline,  the  irregularity 
in  the  size  of  the  grains  of  dirt  they  contain,  and  by  their 
high  refractive  index. 

Flaws,  specks  of  dirt,  or  grease  on  slides  or  cover- 
glasses  may  cause  confusion,  but  may  be  distinguished  in 
the  same  manner.  In  any  case  of  doubt  it  is  well  to 
touch  the  edge  of  the  cover-glass  with  a  needle  whilst 
observing  the  object,  and  in  that  way  it  will  be  seen  that 
the  movement  of  the  object  is  independent  of  the  cor- 
puscle that  was  supposed  to  contain  it. 

In  stained  specimens  there  are  similar  fallacies,  and, 
in  addition,  dirt  from  the  stain,  precipitated  grains  of 
stain,  yeast  cells,  or  other  micro-organisms,  may  be 
present.  It  is  well  in  any  case  of  doubt  to  examine 
some  part  of  the  slide  where  the  stain  has  extended 
beyond  the  blood  film,  and  see  if  the  same  appearances 
are  presented  there. 

In  the  great  majority  of  cases,  if  the  appearances  met 
with  in  normal  blood  have  been  carefully  studied,  par- 
ticularly the  blood  plates  and  the  various  forms  of  de- 
generation of  blood  cells  and  of  stained  precipitates, 
mistakes  are  rare.  Very  rarely  we  do  get  an  appearance 
from  stain  precipitates  deposited  on  a  red  corpuscle  that 
is  difficult  to  distinguish,  and  therefore  we  should  avoid 
diagnosing  malaria  from  a  single  body  believed  to  be  a 


DECEPTIONS  99 

parasite.  It  is  better  in  case  of  doubt  to  look  carefully 
for  a  second  parasite. 

Crescents  should  never  be  diagnosed  on  the  ground  of 
the  shape  only.  A  crescent  always  contains  pigment, 
and  is  longer  than  the  diameter  of  a  red  blood  cor- 
puscle, and  stains  with  basic  stains.  Three  blood  plates 
arranged  in  a  row  may  be  about  the  same  size  and  shape 
as  a  crescent,  but  do  not  contain  pigment  or  stain  like 
a  crescent.  A  transformed  or  altered  crescent  can  be 
mistaken  for  a  quartan  parasite. 

Groups  of  blood  plates  are  sometimes  taken  for  sporu- 
lating  bodies,  and  if  they  surround,  as  they  may,  a  mass 
of  dirt,  the  mistake  is  easily  made.  Even  in  fresh  fluid 
blood  the  peculiar  appearance  of  the  edges  of  blood 
plates  should  prevent  this  mistake,  and  in  stained  speci- 
mens the  manner  in  which  the  blood  plates  stain  will 
enable  them  to  be  recognized. 

Imperfect  fixation  is  a  cause  of  some  errors.  In 
specimens  fixed  by  heat,  or  fixed  in  alcohol  that  has 
absorbed  water,  small  round  bodies,  artificial  vacuoles, 
water  or  air,  are  often  found  in  the  red  cells.  They  may 
be  numerous  in  each  corpuscle,  or  only  one  or  two  may 
be  present.  The  sharp  edge  and  high  refractivity  of 
these  bodies,  as  well  as  the  variation  in  size,  distinguishes 
them  from  parasites. 

Familiarity  with  the  appearances  of  blood  prepared 
in  different  ways  is  necessary,  so  that  these  appearances 
will  cause  no  difficulty,  and  the  recognition  with  certainty 
and  rapidity  of  parasites  will  then  be  easy. 

Leishman-Donovan  bodies,  which  are  described  later, 
are  found  in  kala-azar  in  the  leucocytes  of  the  peripheral 
blood,  but  usually  they  are  very  scanty.  The  large  mono- 
nuclear leucocyte  is  the  variety  in  which  they  are 
ordinarily  found,  but  they  also  occur  in  the  polymorpho- 
nuclear leucocytes.  They  can  be  distinguished  by  the 
double  chromatin  mass — a  smaller  deeply-staining  mass, 
and  a  larger,  more  lightly-stained  one  (vide  Parasites  in 
tissues) . 


100 


CHAPTER    V. 
Parasites  found  in  the  Blood  of  Animals. 

Birds  harbour  two  well-known  species  of  intracorpus- 
cular  parasites — proteosoma  and  halteridium,  and  others 
occur. 

Proteosoma  (Proteosoma  grassi,  fig.  32a)  is  found  in 
comparatively  few  species  of  birds.  Amongst  these  the 
Indian  sparrow  is  the  most  common.  Working  with  this 
parasite,  Major  Ross  first  demonstrated  the  sexual  cycle 
of  the  haemosporidia  in  a  mosquito.  Proteosoma  in  birds 
has  many  resemblances  to  the  malaria  parasite  of  man. 
In  its  earliest  stage  it  is  unpigmented  ;  with  continued 
growth  pigment  appears,  and  as  the  parasite  grows  it 
displaces  the  nucleus  of  the  affected  red  blood  corpuscle, 
and  the  corpuscle  becomes  paler.  All  stages  of  develop- 
ment, from  ring  forms  to  sporulating  forms,  are  found 
in  the  blood  at  the  same  time.  The  gametocytes  are  very 
similar  to  those  of  benign  tertian  malaria.  The  infection 
is  readily  transferred  from  one  bird  to  another  by  inocu- 
lation, and  also  to  other  birds,  such  as  canaries.  It  is 
naturally  transmitted  by  mosquitoes  (Culex  fatigans). 

Halteridium  (Plate  V.,  15)  is  a  common  parasite  of 
many  species  of  birds  ;  it  is  found  in  pigeons,  crows, 
jays,  finches,  parrots,  &c. — often  a  high  percentage  of  the 
birds  of  a  susceptible  species  are  found  to  be  infected. 
The  infection  cannot  be  transferred  from  one  bird  to 
another  by  inoculation,  and  the  method  of  its  transmis- 
sion in  nature  is  doubtful.  Recently  it  has  been  shown 
that  infection  may  be  transmitted  from  one  bird  to 
another   by  a    fly  belonging    to    the    Hippoboscichc    and 


HALTERTDIUM 


IOI 


known  as  Lynchia  maura.  It  was  with  this  parasite 
that  MacCallum  first  demonstrated  the  fertilization  of 
the  macrogamete  by  the  microgamete.  This  parasite  is 
characterized  by  its  peculiar  curved  shape  surrounding 
the  oval  nucleus,  but  not  displacing  it  as  a  rule.  It  does 
not  cause  any  change  in  the  red  corpuscle  which  contains 
it.  Halteridium  resembles  proteosoma  in  producing  pig- 
ment, but  is  easily  distinguished  from  it  by  its  position 
in  the  cell  (fig.  326). 


Fig.  32. — a,  Proteosoma;  /',  halteridium. 


The  forms  of  the  parasite  as  seen  in  the  peripheral 
blood  are  probably  all  sexual  forms. 

If  fresh  fluid  films  of  blood  containing  this  parasite  be 
examined  it  will  be  seen  that  the  halteridia  leave  the  red 
cells  and  go  through  a  similar  series  of  changes  like 
the  "  crescents  "  of  sub-tertian  malaria.  The  males  pro- 
ceed to  flagellate,  whilst  the  females  are  passive  and 
receptive,  and  are  fertilized  by  the  microgametes  with 
the  production  of  an  ookinet.  In  dry  films  stained  by 
Leishman,  or  one  of  its  closely-allied  stains,  the  males 
can  be  easily  distinguished  from  the  females,  the  proto- 
plasm of  the  latter  staining  a  deep  blue,  whilst  in  the 
males  the  protoplasm  is  only  very  faintly  stained. 

The  asexual  phases  of  the  parasite  have  not  been 
clearly  demonstrated. 


102  PIROPLASMA 

Halteridium  has  attracted  a  good  deal  of  attention 
in  view  of  Schaudinn's  remarkable  observations  on  the 
life-cycle  of  Trypanosoma  noctuce,  a  trypanosome  occur- 
ring in  the  blood  of  the  little  owl  (Athene  noctnev). 
According  to  Schaudinn  this  flagellate  attaches  itself  to 
the  red  blood  corpuscle  by  means  of  its  flagellum,  and, 
gradually  penetrating  it,  comes  to  lie  by  the  side  of  the 
nucleus.  In  this  process  the  trypanosome  loses  its 
flagellum  and  undulating  membrane,  and  the  blepharo- 
plast  becomes  closely  applied  to  the  nucleus.  The  flagel- 
late Trypanosoma  nocture,  thus  becomes  transformed  into 
the  halteridium.  The  trypanosome  stages  are  found  at 
night  chiefly  in  the  internal  organs,  while  the  halteri- 
dium phase  is  found  by  day  in  the  peripheral  blood. 
When  fully  grown  the  trypanosome  may  divide  asexually, 
or,  if  the  gametocyte  forms  are  taken  into  the  stomach  of 
the  mosquito  Cnlex  pipiens,  develop  further.  Schaudinn's 
observations  still  await  confirmation.  Some  observers 
are  of  the  opinion  that  probably  both  trypanosomes  and 
halteridia  were  present  in  the  birds,  and  that  Schaudinn 
confused  the  changes  occurring  in  these  two  classes  of 
parasites.  The  frequency  with  which  flagellates  are 
found  in  insects  adds  to  the  doubt  as  to  Schaudinn's 
hypothesis. 

Various  other  parasites  allied  to  those  of  human  malaria 
have  been  found  in  animals.  African  monkeys  harbour 
a  parasite  {Plasmodium  koclii)  closely  resembling  the 
sub-tertian  parasite  of  man,  and  some  species  of  Asiatic 
monkeys,  bats,  and  flying  foxes  harbour  parasites  which 
are  not  unlike  those  of  quartan  malaria. 

In  snakes  a  pigmented  parasite  closely  resembling  a 
large  halteridium  has  been  described,  and  is  known  as 
hcemocystidinm.  Nothing  is  known  of  its  life-history 
or  mode  of  propagation. 

Piroplasmata  (Babesia). — Another  class  of  parasite, 
of  which  several  representatives  have  been  found  in  the 
blood  of  animals,  is  piroplasma.  These  parasites  differ 
from  the  Hcemamceba.',  such  as   the  parasites  of  malaria, 


PIROPLASMA  103 

in  that  they  form  no  pigment,  that  the  nucleolus  does  not 
fragment,  that  division  is  into  two  or  four  only,  and  in 
the  frequency  with  which  extra-corpuscular  forms  are 
found.  All  the  parasites  of  this  class  which  have  so  far 
been  investigated,  have  been  shown  to  have  as  an  inter- 
mediate host  some  species  of  tick  of  the  sub-family 
Ixodina.  The  first  of  these  to  be  discovered  was  Piro- 
plasma bigeminum  (Plate  IV.,  24),  the  cause  of  Texas 
fever  in  cattle.  It  is  transmitted  in  America  by  Rhipi- 
cephalus  annulatus,  and  in  Africa  and  Queensland  bv 
R.  austral  is. 

P.  parvum  causes  Rhodesian  fever  of  cattle.  It  occurs 
in  the  blood  in  bacillar}',  spherical,  and  intermediate 
forms.  It  is  transmitted  by  Rhipicephalus  appeudiculatus. 
An  animal  may  harbour  both  Piroplasma  bigeminum 
and  P.  parvum  at  the  same  time.  P.  bigeminum 
may  be  conveyed  from  one  animal  to  another  bv 
inoculation,  but  similar  experiments  with  P.  parvum 
have  been  negative,  and  the  offspring  of  infected  ticks 
are  not  infective. 

P.  canis  is  the  cause  of  epidemic  jaundice  of  dogs.  It 
is  carried  by  Hcemaphysalis  leachi  in  South  Africa,  and 
Dermaceutor  reticulatus  in  Europe,  and  Rliipicephalus 
sanguineus  in   India. 

Other  species  of  piroplasma  described  are  :  Piroplasma 
ovis,  found  in  sheep  and  carried  by  Rliipicephalus  bursa  ; 
Piroplasma  eqiii,  P.  muris,  and  an  unnamed  species  found 
in  the  monkey  in  Uganda  has  been  described  by  P.  H. 
Ross. 

Experimental  work  has  demonstrated  that  the  parasites 
are  peculiarly  specific,  the  disease  not  being  transmissible 
to  other  animals.  Thus  P.  canis  can  only  reproduce  the 
disease  in  dogs  and  not  in  other  animals.  P.  equi  will 
produce  the  disease  in  horses  and  donkeys,  but  no  other 
animals.  Similarly  with  the  other  known  piroplasmata. 
So  far  piroplasmata,  though  described  in  man  on  several 
occasions,  have  not  been  conclusively  proved  to  occur. 
All  attempts  to  find  them  in  Blackwater  Fever  have  failed. 


104  PIROPLASMA 

An  interesting  fact  is  that  ticks  which  feed  on  an 
infected  animal  are  not  in  themselves  infective,  but  hand 
down  the  infection  to  their  offspring.  These,  sometimes 
as  larvae,  in  other  cases  not  till  they  become  nymphs  or 
adults,  are  able  to  convey  the  infection  to  the  animal 
on  which  they  feed,  provided  that  animal  is  susceptible 
to  the  particular  piroplasma  involved. 

Development  of   Piroplasma. 

Asexual  Cycle. — This  parasite  has  a  free  and  an  intra- 
corpuscular  stage  in  its  asexual  life-history.  In  the  free 
stage  they  are  pyriform  in  shape,  and  in  the  intracorpus- 
cular  at  first  rounded,  afterwards  dividing  into  two  or 
more  pyriform  bodies  by  a  process  of  budding.  The 
newly-found  parasites  are  at  first  connected  together  by 
a  thin  process,  but  finally  become  separated,  and  escaping 


Fig.    33. 

from  the  corpuscle  invade  other  corpuscles.  They  may 
enter  and  leave  two  or  three  corpuscles  before  again 
becoming  rounded  and  undergoing  division  ;  ultimately, 
however,  the  process  of  division  is  undergone,  and  so  the 
cycle  is  repeated. 

Sexual  Stage. — Koch  has  described  developmental  forms 
of  P.  bigeminum  in  Rhipicephalus  auslialis,  R.  evertsi  and 
Hyaldmma  cegypticum,  and  Christophers  has  demonstrated 
similar  forms  of  Piroplasma  canis  in  Rhipicephalus  san- 
guineus. From  these  observations  it  appears  that  in  the 
stomach  of  the  tick  the  parasite  escapes  from  the  red 
blood  corpuscle  and  becomes  elongated,  one  of  its 
chromatin  masses  passing  to  the  blunt  (?  anterior)  end, 
forming  a  projection — the  other  chromatin  mass  remain- 
ing in  the  middle  of  the  parasite.     Next  radial  processes 


H^MOGREGAKINA  105 

arise  near  the  projection,  the  other  end  of  the  parasite 
becomes  pointed,  and  the  general  contour  of  the  parasite 
becomes  angular.  After  the  second  day  couples  of  these 
forms  are  seen  apparently  connected  together,  a  process 
of  copulation.  The  male  element,  after  giving  up  its 
chromatin,  is  thrown  off  or  stretched  out  over  the  en- 
larged fertilized  female.  The  fertilized  element  after 
becoming  round,  oval,  or  eventually  club-shaped,  leaves 
the  gut  and  passes  to  the  ovary.  In  the  ova  of  infected 
ticks  large  pear-shaped  bodies  are  described.  Christophers 
also  states  that  he  has  found  further  developmental  forms 
in  the  cells  of  the  gut  of  unfed  nymphs  reared  from 
infected  mothers,  and  has  traced  them  to  the  salivary 
glands  of  nymphs  of  the  second  generation,  which  were 
about  to  become  adults. 

H^EMOGREGARINA. — The  haemogregarines  are  unpig- 
mented  parasites,  occurring  both  as  intracorpuscular  forms, 
in  which  they  are  bent  upon  themselves  so  as  to  form 
a  tail  (Plate  V.,  19),  and  as  free  vermicular  forms  in  the 
blood  plasma  (Plate  V.,  17  and  20).  They  are  non-pig- 
mented,  and  in  stained  specimens  the  nucleus  stains  well, 
and  contains  abundant  chromatin  as  numerous  granules 
sometimes  arranged  in  radiating  lines.  Sporulation  takes 
place  not  in  the  blood,  but  in  certain  cells  of  the  viscera, 
usually  of  the  liver. 

The  parasites  are  common  in  the  blood  of  cold- 
blooded vertebrates.  The  effect  on  the  red  corpuscle 
varies.  Some  species  displace  the  nucleus,  some  do  not. 
At  least  one  species  causes  the  formation  of  granules 
which  stain  like  the  Schuffner's  dots  in  the  human  cor- 
puscle containing  the  parasite  of  benign  tertian  malaria 
(Plate  V.,  21).  Several  species  have  recently  been  found 
in  the  blood  of  mammals.  None  have  been  found  in 
human   blood. 

The  first  of  these  mammalian  haemogregarines  was 
discovered  by  Bentley  in  the  leucocytes  of  dogs  in  Assam, 
and  described  by  James  as  Lcucocytozoon  canis  (correctly 
H.  canis).     Soon  afterwards  similar   parasites  were   des- 


JO'S 


H.EMOGREGARINA 


cribed  by  Balfour  as  occurring  in  the  red  blood  cor- 
puscles of  the  jerboa,  Jaculus  goudoni  (fig.  346),  and  by 
Christophers  in  the  Indian  field-rat,  Gerbillus  indicus. 
Later,  Patton  described  another  haamogregarine  in  the 
leucocytes  of  the  Kathiawar  squirrel,  Funambulus  pcu- 
nantii,  and  Balfour  a  similar  parasite  in  the  leucocytes 
of  the  rat  (Mits  decumanus). 


Fig.  34a. — Hsemogregarine  in  Reptilian  blood. 


Fig.  34A — Hsemogregarine  in  Mammalian  blood. 


H.  canis  occurs  in  the  polymorphonuclear  leucocytes 
only.  The  parasite  is  enclosed  in  a  capsule  which  is  not 
easily  penetrated  by  the  stain.  It  is  an  oblong  body 
rounded  at  the  ends.  The  contents  are  the  haemo- 
gregarine,  which  is  sharply  bent  on  itself,  so  that  the 
nucleus  is  frequently  horse-shoe  shaped. 

The  development  of  this  parasite  has  been  studied  by 
Christophers.  It  has  been  found  to  reproduce  itself  by 
encystment  in  the  bone-marrow  of  the  host,  there  form- 
ing merozoites  (fig.  35).  He  also  found  that  the  sexual 
development  of  the  parasite  took  place  in  the  dog  tick 
(R.  sanguineus).  When  taken  into  the  gut  of  ticks  the 
parasite   escapes    from    the    capsule    and    shows    active 


\ 


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Xi) 


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f 


\y. 


U.  v#  flK 


♦  ♦ 


Fig.  35. 


t«  ^.  106, 


HLEMOGREGARINA  107 

vermicular  movements.  Within  twenty-four  hours  these, 
associated  in  pairs,  have  lodged  themselves  in  the  large 
cells  of  the  gut.  On  the  second  day  conjugation  between 
two  similar  individuals  takes  place,  and  a  globular  body 
is  formed  containing  a  single  large  homogeneous  mass 
of  chromatin.  This  chromatin  mass  spreads  out  to  form 
a  reticulum,  and  the  protoplasm  increases  in  amount. 
The  chromatin  collects  at  the  periphery  into  irregular 
star-shaped  masses,  and  on  the  third  or  fourth  day  the 
whole  body  splits  up  to  form  eleven  to  fourteen  sporo- 
zoites.  When  set  free  the  sporozoites  are  in  the  lumen 
of  the  gut.  The  method  of  re-entry  in  the  dog  has  not 
been  discovered. 

H.  balfouri  occurs  in  the  red  blood  corpuscles  of  the 
jerboa  or  desert-rat.  As  seen  in  stained  specimens  it 
is  a  slightly-curved  body,  with  rounded  ends  lying  either 
apparently  free  or  in  the  remains  of  a  red  blood  cor- 
puscle. It  is  believed  that  the  free  forms  owe  their  con- 
dition to  the  destruction  of  the  red  blood  cell  which 
contained  them.  A  long  oval  nucleus,  situated  about 
the  centre  of  the  parasite,  is  present,  and  occasionally 
a  few  dots  of  chromatin  in  the  pale  polar  areas. 

The  stage  of  schizogony  has  been  observed  by  Balfour 
to  take  place  in  the  liver  cells  of  the  host  (fig.  35),  and  is 
similar  to  that  described  in  H.  can  is. 

H.  gerbilli  is  a  parasite  similar  to  the  preceding,  occur- 
ring in  the  red  blood  corpuscles  in  the  Indian  field-rat. 

The  stage  of  sporogony  occurs  in  the  louse.  The 
complete  life-cycle  has  not  been  discovered. 

H.  fiinambuli  is  a  parasite  occurring  in  the  large 
mononuclear  leucocytes  of  the  palm  squirrel.  It  differs 
from  H.  canis  in  the  absence  of  a  capsule  surrounding 
the  parasite.  A  similar  parasite  has  been  found  by 
Balfour  in  the  Norway  rat. 

Gregarines. — These  protozoa  are  characterized  by 
certain  peculiarities  in  the  reproduction.  They  are  not 
known  to  occur  in  any  true  vertebrate,  though  they  are 
found  in  amphioxus.      They  are  pre-eminently  parasites 


io3 


H.-EMOGRKGAKIXA 


of  arthropods,  but   occur  commonly   in    worms.      They 
are  very  rare  in  molluscs. 

The  gregarine  is  primarily  a  parasite  of  the  digestive 
tract,  in  which  in  the  earliest  stages  it  is  attached  to  or 
contained  in  an  epithelial  cell  of  the  gut.  When  the  host 
cell  is  used  up  the  parasite  may  remain  free  in  the  gut  or 
may  penetrate  into  the  body  cavity.  When  special  por- 
tions of  the  ccelom  are  separated  for  a  particular  func- 
tion, as  in  the  case  of  the  vesiculae  seminales  of  the 
earthworm,  the  parasites  may  be  found  in  these  organs 


Fig.  35a. — Development  of  Gregarines.  a,  The  two  sporonls  in  a  common 
cyst ;  b,  various  stages  of  nuclear  division  in  the  sporon's ;  c,  commencing 
formation  of  gametes  by  a  process  of  sporulation  ;  d,  union  of  gametes  in  pairs 
to  form  zygotes  ;  e,  stages  in  the  division  of  the  nuclei  of  the  zygotes  ;  /,  cyst 
with  ripe  spores,  each  containing  eight  sporozoites. 

Reproduction  always  takes  place  by  sporogony,  except 
in  the  small  sub-order  Schizogregarinaj.  Hence  Grega- 
rinae  are  divided  up  into  two  sub-orders — Schizogregarinae 
and  Eugregarinae.  The  Eugregarinae  are  further  divided 
into  Cephalina  and  Acephalina. 

The  Cephalina  have  a  well-defined  epimerite  or  fixative 


H^EMOGREGARINA  IO9 

organ,  and  the  body  is  usually  divided  up  by  septa.  An 
example  is  the  gregarine  found  in  the  gut  of  the  meal- 
worm. 

The  Acephalina  are  those  which  have  no  fixative  organ 
or  epimerite,  and  the  body  is  not  divided  up  by  septa. 
The  best-known  examples  are  the  Monocystis  magna  and 
Monocystis  agilis  found  in  the  vesicular  seminales  of 
earth-worms. 

The  body  form  of  gregarines  is  always  well  defined, 
never  amoeboid,  and  varies  from  a  sphere  or  ovoid 
to  an  elongated  wormlike  shape.  The  body  is  com- 
posed of  ectoplasm  and  endoplasm,  in  the  latter  of 
which  is  the  large  single  ovoid  nucleus. 

Reproduction  is  by  sporogony.  Two  full-grown 
gregarines  come  together,  become  rounded  and  develop 
a  cyst  wall  around  themselves.  The  nuclei  become  frag- 
mented, and  around  these  fragments  of  nuclei  segmenta- 
tion of  the  protoplasm  takes  place  to  form  gametes. 

Conjugation  of  gametes  then  takes  place  to  form 
zygotes,  each  of  which  becomes  encysted  and  develops 
within  itself  eight  sporozoites  (fig.  35^). 

These  sporozoites  when  introduced  into  a  fresh  host 
develop  into  adult  gregarines. 

Zoological  Position  of  the  Malaria  Parasites. 

Protozoa 

! 

Class  Sporozoa 


Sub-class  Telosporidia  Neosporidia 


Order  (1)   Gregarinoidea  Hcemosporidia  Coccidiidea 

I 
Genera  : 
(1)   Plasmodium    (Hcem-     (2)  Hamo°regarina      (3)  Piroplasma      (4)  Halteri- 
amccba)  dium  (?) 

/    P.    vivax    (Benign     tertian      H.  ranarian  P.   bigeminitm 

J  parasite)  H.  gerbilti  P.  pa?-v?im 

P.malarius  (Quartan  parasite)     H.  half  our  i  P.  ovis 

P.   falciparum    (Sub-tertian     H.  canis  P.  equi 

parasite)  H.  funambuli  P.  canis 

P.    ptcvcox    (Proteosoma)  &c.  &C. 

\  P.  kochi  (found  in'monkeys) 
&c. 


■8  J 


no 


CHAPTER    VI. 
Parasites  found  in  Blood  Plasma. 

The  most  important  animal  parasites  found  in  the 
plasma  of  the  peripheral  blood  are  trypanosomes  and 
spirochaetae  belonging  to  the  class  Mastigophora,  and 
embryos  of  various  filaria,  nematode  worms. 

In  some  of  the  lower  animals  haemogregarines  are  found 
during  their  motile  stage,  and  the  piroplasma  pass  a  short 
period  of  their  existence  free  and  may  be  found  in  the 
plasma.  The  haemamcebidaa  are  not  found  in  the 
plasma,  though  for  a  brief  period  the  "  spores  "  must  be 
free.  As  stated  in  the  preceding  chapter,  according  to 
Schaudinn,  the  halteridium  has  a  free  stage  in  the  plasma. 

In  freshly-shed  blood,  trypanosomes  are  readily  seen 
as  actively  motile,  worm-like  bodies  darting  about  be- 
tween the  blood  corpuscles.  Their  movements  are  so 
rapid  and  they  are  so  transparent  that  it  is  difficult  to 
make  out  their  form  clearly  in  the  living  condition. 

The  largest  trypanosomes  are  found  in  fish,  both  fresh 
and  salt  water.  They  can  readily  be  demonstrated  in 
small  fish  by  cutting  off  their  heads  and  making  a 
sme;ir  on  the  slide  with  the  cut  surface.  These  smears 
can  be  examined  fresh  by  placing  a  cover-glass  on  the 
top  of  the  exuded  fluid,  or  the  films  can  be  dried  and 
stained. 

Trypanosomes  are  found  in  some  birds.  The  method 
of  examination  of  the  blood  for  these  is  the  same  as  that 
required  for  the  haemosporidia.  If  a  small  bird  is  to  be 
examined  it  is  held  in  the  palm  of  the  left  hand  and  one 
leg  is  allowed  to  protrude  between  the  fingers.  A  needle 
is  then  inserted  deeply  into  the  vascular  pad  surrounding 


TRYPANOSOMA  1 1 1 

the  root  of  the  claw  and  left  there  for  half  a  minute.  On 
squeezing  the  leg  so  as  to  force  the  blood  towards  the 
claw  the  blood  will  exude  in  drops,  and  films  can  be 
made  as  with  human  blood.  With  larger  birds  an 
assistant  is  necessary  to  hold  the  bird,  and  the  bird  should 
be  wrapped  in  a  thick  cloth  for  the  protection  of  the 
assistant  and  operator. 

The  trypanosomes  that  have  attracted  most  attention 
are  those  of  the  mammalia.  Many  species  are  known ; 
they  closely  resemble  each  other  in  their  appearance,  but 
differ  in  size,  shape,  and  motility  to  some  extent  ;  also  in 
the  positions  they  assume  and  the  way  in  which  they 
stain  (Plate  I.,  a,  b,  c,  d,  c).  They  may  be  differentiated 
by  their  pathogenic  action.  By  inoculating  a  series  of 
animals  with  the  blood  it  will  then  be  found  which 
animals  are  immune  and  which  are  susceptible.  For 
such  inoculation  the  blood  should  be  used  at  once,  but 
if  this  is  impossible  then  it  may  be  mixed  with  some 
fluid  that  will  prevent  coagulation.  Sodium  citrate  solu- 
tion, 10  per  cent.,  may  be  used,  and  the  blood  should 
be  diluted  with  one-twelfth  of  this  solution.  Others  use 
a  weaker  solution  of  citrate  of  soda,  i  per  cent.,  and 
dilute  the  blood  more  freely.  Injection  of  such  diluted 
blood  into  the  subcutaneous  tissues  will,  in  the  majority 
of  cases,  lead  to  infection  with  trypanosomata  of  the 
animal  which  has  been  injected  if  it  is  susceptible. 

Every  mammalian  trypanosome,  so  far  as  is  known, 
belongs,  with  the  exception  of  T.  cruzi,  to  one  of  five  types 
(Plate  I.).     These  types  are  : — 

(A)  T.  lewisii.  The  antiflagellar  end  is  sharply  pointed, 
the  centrosome  usually  at  some  little  distance  from  the 
end  of  the  body,  and  the  nucleus  is  always  in  the  posterior 
half  of  the  body.     Size  25  to  30  //,  (Plate  I.,  b). 

(B)  T.  gambiense.  The  antiflagellar  end  is  usually 
bluntly  rounded,  the  centrosome  close  to  that  end,  and 
the  nucleus  at  the  middle  of  the  body.  Size  20  to  25  pu 
(Plate  I.,  d). 

(C)  T.    naninu.     Characterized  by  its  small  size,  being 


112  TRYPANOSOMIASIS 

only  about  14  /x  in  length.  The  centrosome  is  small  and 
the  nucleus  is  round  and  in  the  centre  of  the  body 
(Plate  I.,  c). 

(D)  T.  theileri.  Characterized  by  its  large  size,  being 
some  65  /jl  in  length  (Plate  I.,  e). 

(E)  T.  tliiiiorplwu.  The  important  characteristics  of  this 
type  are  the  broad  body,  the  short  free  portion  of  the 
flagellum,  the  close  application  of  the  undulating 
membrane  to  the  body  and  the  presence  of  a  vacuole 
between  the  centrosome  and  nucleus  (Plate  I.,  a). 

The  more  important  of  the  trypanosomata  are  :  — 

(A)  (1)  Those  found  in  a  large  proportion  of  the  rats  in 
both  tropical  and  temperate  climates.  These  are  non- 
pathogenic to  full-grown  rats,  and  all  other  animals 
experimented  on  are  insusceptible  to  the  infection  ('/'. 
lewisi  (Plate  I.,  b). 

(B)  (1)  Nagana,  or  "Tsetse-fly  Disease."  The  trv- 
panosoma  of  this  disease  (T.  brucei)  can  be  inoculated  into 
a  large  number  of  wild  and  domesticated  animals,  but 
man  is  insusceptible.  To  cattle,  horses,  donkeys,  dogs, 
guinea-pigs,  rats,  &c,  this  parasite  is  pathogenic,  but  the 
time  required  to  cause  death  varies  greatly  in  these 
animals.  Wild  game,  and  particularly  the  buffalo,  harbour 
the  parasite,  which  appears  to  be  harmless  to  them.  It 
is  carried  from  animal  to  animal  by  biting  flies  belonging 
to  the  genus  Glossina,  usually  by  G.  morsitans. 

(2)  Surra  (7\  evansi).  A  disease  fatal  to  horses  ;  cattle 
often  recover.  It  occurs  in  India,  Philippines,  <&c.  It 
is  carried  by  various  biting  flies,  such  as  Stomoxys 
calcitrant,  some  of  the  tabanidas,  hasmatobia,  &c. 

In  the  living  condition  it  can  be  distinguished  from 
T.  brucei  by  its  greater  activity,  as  it  not  only  moves  but 
actually  progresses. 

(3)  Dourine  (T.  equiperdum).  A  disease  chiefly  affect- 
ing mares  and  stallions  and  found  in  Southern  Europe, 
Northern  Africa  and  Chili.  It  is  a  disease  transmitted  bv 
coitus  and  characterized  chiefly  by  a  purulent  discharge 
from  the  genitals  and  great  oedema  of  the  abdomen.     It 


Plate  I. 


O 


% 


Bale  &  Daniels  son,  iM  del.et  liLh. 


TRYPANOSOMIASIS  1 1  3 

is  accompanied  by  fever  and  great  emaciation  and  usually 
proves  fatal.  The  parasites  are  found  abundantly  in  the 
discharges  and  scantily  in  the  blood.  Rats,  dogs,  rabbits, 
&c,  are  susceptible,  whilst  monkeys,  cattle,  sheep  and 
goats  are  refractory. 

(4)  Mal  DE  Caderas  (T.  cqiiiiium).  A  disease  of  horses 
in  South  America,  notably  in  Venezuela.  The  parasite  is 
characterized  by  having  a  very  small  centrosome.  The 
natural  host  of  the  parasite  is  believed  to  be  the  capybara 
— a  large  water  vole.  The  carrier  is  probably  a  stomoxys. 
The  disease  can  be  inoculated  into  most  animals,  but 
cattle  are  immune. 

(5)  T.  gambiense  (Plate  I.)  Man  is  insusceptible 
to  all  the  above-mentioned  trypanosomes,  but  in  Africa, 
on  the  West  Coast  and  throughout  a  large  part  of 
Central  Africa,  another  species  has  been  found  in  man. 
The  parasites  are  found  in  small  numbers  in  the  blood 
in  most  cases,  particularly  in  chronic  cases,  but 
can  be  found  in  larger  numbers  in  fluid  drawn  with 
an  aspirating  needle  from  a  lymphatic  gland.  The 
symptoms  are  constant  irregular  pyrexia,  enlarged  lym- 
phatic glands,  usually  in  the  neck  or  above  the  clavicle, 
erythematous  rash.  Enlargement  of  spleen  and  liver 
have  been  noted.  Monkeys  are  susceptible,  and,  with 
some  strains  dogs,  white  rats  and  guinea-pigs.  After  a 
longer  or  shorter  period  in  man  grave  cerebral  symptoms 
supervene,  usually  of  the  lethargic  condition  known  as 
"  sleeping  sickness,"  and  the  patient  dies.  In  this  stage 
of  the  disease  trypanosomes  are  found  in  the  cerebro- 
spinal fluid.  They  are  not  numerous,  and  it  is  necessary 
to  centrifugalize  the  fluid  to  demonstrate  them. 

(C)  T.  nanum  is  found  in  cattle  in  the  Soudan, 
causing  in  them  fever,  emaciation,  &c.  The  carrier  is 
believed  to  be  stomoxys. 

(D)  T.  theileri.  South  Africa.  Cattle  only  are  sus- 
ceptible. No  other  animal  has  yet  been  inoculated 
successfully.  It  is  one  of  the  largest  of  the  trypanosomes 
found  in  mammals.  It  is  believed  to  be  transmitted  by 
H ippobosca  rufipes. 


114  TRYPANOSOMIASIS 

(E)  T.  DIMORPHON  occurs  in  horses  in  Africa,  especially 
on  the  Gambia.  The  disease  produced  is  like  "  Nagana" 
but  more  chronic.  Various  animals  are  susceptible. 
The  parasite  occurs  in  several  forms. 

T.  CRUZI.  This  trypanosome,  or  rather  schizotrypanum, 
was  discovered  in  the  blood  of  a  child  in  Brazil  who  was 
suffering  from  irregular  fever,  progressive  anaemia,  and 
enlargement  of  lymphatic  glands. 

The  carrier  is  believed  to  be  a  bug — Conorhinus  sciii^ni- 
suga — belonging  to  the  Keduviidae. 

More  recent  work  by  Chagas  on  this  parasite  has  shown 
that  it  differs  in  several  respects  from  the  ordinary 
trypanosomes.  Instead  of  multiplying  by  longitudinal 
fission  in  the  peripheral  blood,  it  splits  up  into  eight 
merozoites  within  its  limiting  membrane  in  the  capillarie> 
of  the  lung.  In  consequence  of  its  mode  of  multiplica- 
tion it  has  been  named  by  Chagas — Schizotrypanum. 
The  bug  which  is  the  carrier  acts  as  a  true  host,  for  after 
feeding  on  an  infected  man  or  animal,  it  is  not  capable  of 
infecting  another  till  eight  days  have  passed,  but  after 
that  remains  infective  for  an  indefinite  period. 

For  the  examination  of  blood  for  trypanosomes,  films 
prepared  as  for  malaria  are  the  best,  as  the  parasite  will 
then  be  seen  undistorted.  When  the  parasites  are  scanty 
and  for  purely  diagnostic  purposes,  thicker  films  de- 
colorized by  the  action  of  water  may  be  used,  but  a  good 
deal  of  distortion  results.  In  some  infections  the  parasites 
can  only  be  found  by  injecting  a  highly-susceptible 
animal  with  the  blood  of  a  suspected  case,  a-,  a  large 
infection  may  then  result  in  the  animal  which  has  been 
injected.  This  proceeding  is  necessary  in  many  cases  of 
donrine.  In  centrifugalized  blood  the  parasites  accumu- 
late in  the  upper  part  of  the  mass  of  red  coipuscles  and 
can  be  found  there  more  readily  than  by  the  ordinary 
method. 

Tvpanosomes  stain  rather  feebly  with  most  ba^ic 
stains,  luematoxylin,  methylene  blue,  &c.  A  stronger 
basic  stain,  such  as   carbol  fuchsin,  should  therefore  be 


STAINING   TRYPANOSOMES  1 15 

used.  Clearer  specimens  are  obtained  by  diluting  the 
stain  with  three  parts  of  water  and  leaving  to  stain  for  ten 
minutes. 

Good  results  can  also  be  obtained  by  overstaining  with 
this  stain  and  then  decolorizing  with  ^  per  cent,  solution 
of  glacial  acetic  acid  in  water,  but  the  parasite  is  often 
swollen  and  distorted,  though  quite  recognizable. 

Leishman's  stain,  used  as  for  other  blood  work,  gives 
excellent  results  with  fresh  specimens  and  shows  well 
the  various  points  in  the  structure.  The  body  is  elon- 
gated and  one  extremity  is  bluntly  truncated,  whilst 
the  other  is  prolonged  into  a  long  flagellum.  In  addi- 
tion there  is  attached  to  the  body  of  the  parasite  and 
running  its  whole  length  an  undulating  membrane  fre- 
quently thrown  into  folds.  The  flagellum  is  continued 
throughout  the  body,  running  along  the  free  edge  of  the 
undulating  membrane  and  ciosely  following  its  sinuosities. 
Slightly  posterior  to  the  termination  of  the  flagellum  is 
a  deeply-staining  nodule — the  centrosome.  About  the 
middle  of  the  body  is  a  rounded  mass,  larger  but  less 
defined — the  nucleus.  In  fission  forms  the  centrosome 
first  divides,  then,  successively,  undulating  membrane, 
nucleus  and  protoplasm  (fig.  36).  The  flagellum  does  not 
divide  but  remains  attached  to  one  of  the  resulting 
individuals,  whilst  the  other  develops  a  new  flagellum. 
The  protoplasm  with  Leishman's  stain  is  blue.  The 
centrosome,  nucleus  and  flagellum  are  red. 

Multiplication  is  by  fission.  These  fission  forms 
are  rarely  found  in  the  peripheral  blood  in  man.  Occa- 
sionally there  are  two  flagella,  with  no  signs  of  fission  in 
centrosome  or  nucleus. 

An  interesting  phenomenon  frequently  associated  with 
trypanosome  infections  is  that  known  as  auto-agglutina- 
tion. When  a  fresh  living  film  of  the  infected  blood  is 
examined  it  will  frequently  be  found  that  the  red  blood 
cells  tend  to  run  together  into  clumps,  and  as  this 
phenomenon  has  only  rarely  been  observed  with  other 
infections  it  appears  to  have  some  value  as  a  diagnostic 
sign  in  trypanosomiasis. 


n6 


MULTIPLICATION    OF  TRYPANOSOMES 


The  human  trypanosome  is  carried  by  G.  palpalis, 
but  other  species  of  glossina  are  also  suspected  of  act- 
ing as  carriers. 

The  mode  of  transmission  may  be  direct,  the  trypano- 
somes  being  taken  from  any  infected  animal,  and  without 
any  further  development  in  the  fly  enter  the  next  animal 
bitten.     This  only  occurs  if  the  fly  after  feeding  on  an  in- 


Fig.   36 


fected  animal  proceeds  to  feed  again  almost  immediately 
on  another  animal. 

It  has  now  been  shown  that  a  glossina  very  shortly 
after  feeding  on  an  infected  animal  becomes  incapable 
of  transmitting  the   infection.      If,    however,    the    fly    be 


SPIROCPLETA  117 

kept  alive,  after  a  period  of  eighteen  days  or  so  it  again 
becomes  infective,  thus  pointing  to  some  cycle  of 
development  taking  place  in  the  fly.  Nothing  is  known 
definitely  of  the  phases  of  this  supposed  developmental 
cycle.  No  sexual  phase  has  been  observed  in  the  try- 
panosomes.  Further  work  is  much  required  on  this 
subject. 

In  three  cases  of  trypanosomiasis  in  man,  in  which  the 
infection  seemed  to  have  been  contracted  in  Rhodesia,  it 
was  noted  that  not  only  were  the  parasites  much  more 
virulent  than  T.  gambiense  to  man  and  to  lower  animals 
but  also  that  in  one  case  the  form  of  some  of  the  para- 
sites was  different,  the  nucleus  being  situated  close  to  the 
micronucleus,  and  in  some  instances  even  between  the 
micronucleus  and  the  antiflagellar  end.  The  parasite 
in  man  in  the  Rhodesian  cases  seems  to  be  very  resistant 
to  atoxyl,  though  this  peculiarity  is  not  retained  when 
the  parasites  are  inoculated  into  lower  animals.  Stephens 
suggests  that  in  his  case  the  morphological  differences 
are  sufficient  to  be  considered  specific,  and  describes  the 
parasite  as  T.  rhodesiense. 

Spiroch^eta. 

The  spirochaeta  of  relapsing  fever — Spirochceta  recur- 
rentis  (fig.  37) — is  now  generally  believed  to  belong  to 
the  Mastigophora.  They  can  be  seen  in  fluid  blood  films 
made  as  for  malarial  blood.  The  organisms  are  very 
transparent  and  can  only  be  seen  in  fresh  fluid  prepara- 
tions with  the  diaphragm  nearly  closed.  They  are  then 
seen  as  fine,  transparent,  thread-like  bodies,  which  are  in 
active  movement  and  coil  and  uncoil  themselves.  They 
are  also  seen  in  the  corkscrew-like  forms  which  are  com- 
monly drawn  as  representing  them. 

Dried  films  are  best  thin.  In  such  films  the  spirochetal 
are  seen  in  the  undulating  form  (tig.  37/').  In  thicker 
films  they  appear  more  frequently  coiled  up  (fig.  37^7). 
The  spirochastas  stain  with  all  basic  stains,  but  not 
intensely,  and  are  best  demonstrated  by  the  use  of  the 


u8 


SPIKOCH/KTA 


stronger  basic  stains,  such  as  carbol  fuchsin  diluted  i  to  3 
Of  water  (Plate  VI.,  23). 

They  stain  well  by  Leishman's  method  or  with  Giemsa's. 
Ordinarily  no  definite  structure  can  be  made  out. 

The  disease  can  be  reproduced  in  monkeys,  and  less 
readily  in  rats.  African  tick  fever  has  been  shown  by 
Ross  and  Milne  to  be  caused  by  a  similar  spirochaeta — 


© 


© 


S.  duttoni.  1  n  appearance  it  closely  resembles  S.  recurrentis, 
but  is  more  readily  inoculated  into  lower  animals,  rats, 
guinea-pigs,  <&c.  The  pathogenicity  of  the  two  parasites 
differs,  and  infection  with  one  does  not  affect  the  sus- 
ceptibility to  infection  by  the  other  spirochaetae. 

In  man  there  is  leucocytosis  and  marked  relative  in- 
crease of  the  polymorphonuclear  leucocytes.  This  in- 
crease persists  to  some  extent  in  the  periods  of  apyrexia, 


SPIROCH.ETA 


119 


so  that  a  differential  count  of  the  leucocytes  may  exclude 
malaria. 

The  spirochaeta  shows  no  signs  of  longitudinal  division 
in  the  blood,  and  in  human  blood  has  no  tendency  to 
great  variation  in  length.  It  is  found  in  the  plasma, 
never  in  the  red  blood  corpuscles.  The  spleen  enlarges, 
and  in  fatal  cases  spirochaetae  are  found  in  large  numbers  in 
that  organ.  The  organisms  are  found  in  greatest  number 
during  the  first  pyrexial  period.  In  the  apyrexial  period 
they  are  not  to  be  found,  and  in  the  subsequent  pyrexial 


Fig.  38. — a,  Single  trypanosome  much  enlarged ;  b,  stage  of  fission  ; 
e,  parasites  still  attached  by  posterior  ends  ;  d,  same,  both  parasites  com- 
mencing to  divide,  nucleus  and  blepharoplast  divided  ;  e,  resultant  stage  of 
division  ;  f,  one  of  the  four  spirilla  into  which  e  has  divided. 

attack  they  are  found  in  smaller  numbers  than  in  the 
primary  attack.  In  some  cases  the  disease  passes  on 
into  a  chronic  condition  of  irregular  pyrexia — secondary 
fever ;  it  is  exceptional  to  find  the  parasites  during  that 
period. 

Spirochaetae  are  found  in  the  mouth  and  sometimes  in 
expectoration  and  fasces.  In  various  syphilitic  lesions, 
S.  pallida,  in  yaws,  S.  pertenius,  in  sclerosing  granuloma 
and  in  many  ulcers  spirochaetae  are  found.  The  best 
known  of  these  is  S.  pallida,  found  in   syphilis.      Spiro- 


120  LEISHMAN-DONOVAN    BODIES 

chaetas  arc  found  in  the  blood  of  many  of  the  lower 
animals. 

A  convenient  way  of  demonstrating  S.  pallida  is  by  the 

Indian  ink  method.  The  surface  of  the  lesion  is  scraped 
until  a  drop  of  serum  is  obtained.  This  is  transferred  to 
a  slide  and  there  mixed  with  two  drops  of  Indian  ink 
(Gunther's).  The  mixture  is  then  spread  with  another 
slide  as  in  making  a  blood  film.  The  film  is  allowed  to 
dry  and  can  be  examined  with  the  oil  immersion  lens 
without  applying  a  cover  slip.  The  spirochaetae  are  seen 
as  bright  spirals  on  a  dark  brown  field.  This  method 
can  also  be  used  for  the  detection  of  spirochetal  in 
sputum. 

Schaudinn  believed  that  the  spirochaetae  are  closely 
related  to  the  trypanosomes,  and  therefore  belong  to  the 
uwstigopliora  or  flagclhita.  He  considered  them  to  result 
from  the  repeated  longitudinal  division  of  the  trypano- 
somes, the  nucleus  and  centrosome  becoming  both 
elongated  and  attenuated. (fig-  3^). 

Leishmax-Doxovax  Bodies. 

These  parasites  are  found  sparingly  in  the  blood,  in 
the  leucocytes,  either  in  the  large  mononuclear  or  in 
the  polymorphonuclear.  They  are  said  to  become  very 
numerous  just  before  death. 

Possibly  a  free  flagellate  form  may,  in  time,  be  found 
in  the  plasma,  as  in  cultures  the  bodies  develop  flagella. 
These  flagellate  forms  have  not  been  observed  in  man. 

The  bodies  are  found  in  the  spleen,  liver,  lymphatic 
glands,  lungs  and  submucosa,  and  occur  in  these  situa- 
tions, particularly  in  the  spleen  and  liver,  in  enormous 
numbers.  They  are  contained  in  the  endothelial  cells, 
and  in  masses  imbedded  in  a  hyaline  matrix  between 
the  cells. 

They  can  be  observed  in  smears  from  the  organs  taken 
alter  death,  or  by  puncture  and  aspiration,  with  a  hypo- 
dermic syringe,  of  the  spleen  or  liver.  Fatal  accidents 
have  resulted  from  puncture  of  the  spken,  and  punctures 


Late    II. 

/;. — Cryptococcus. 


a — Leishman-Donovan  bodies. 


Fig.  39. 

Bale  &  Damelsson,Lt.i  del.et  lith. 


LEJSHMAN-DOXOVAN    BODIES  121 

of  the  liver  should  therefore  be  made,  though  the  para- 
sites are  not  found  so  readily  in  the  fluid  drawn  from 
the  liver.  In  some  cases  they  may  be  obtained  by  punc- 
ture of  the  superficial  lymphatic  glands.     (Cochran). 

A  large  all-glass  syringe  is  convenient  for  the  pur- 
pose. The  needle  should  not  be  too  fine.  The  skin 
must  be  carefully  sterilized  over  the  selected  place  and 
the  syringe  and  needle  sterilized  dry.  The  needle  should 
be  plunged  right  into  the  liver,  so  that  it  moves  with  the 
movements  of  the  liver.  It  should  be  rotated  and  with- 
drawn a  little,  but  still  kept  in  the  liver  before  aspiration. 
The  aspiration  should  not  be  too  forcible. 

If  much  blood  comes  this  will  so  dilute  the  fluid  con- 
taining the  Leishman-Donovan  bodies  that  a  prolonged 
search  may  be  necessary  to  find  them. 

They  swell  up  and  break  down  on  the  addition  of 
water,  so  that  thick  decolorized  blood  films  cannot  be 
used.  It  is  for  this  same  reason  that  the  syringe  used 
for  aspirating  must  be  dry. 

The  bodies  are  small,  round,  or  oval  masses  of  proto- 
plasm, which  stain  faintly  with  basic  stains,  and  contain 
two  chromatin  masses,  which  stain  deeply  with  ordinary 
basic  stains,  and  with  the  polychrome  methyl  blue  a 
deep  red.  These  two  chromatin  masses  are  unequal  in 
size.  The  larger  is  oval,  is  situated  to  one  side  of  the 
parasite,  and  stains  with  Leishman  a  decided  red,  but 
not  very  deeply.  The  smaller  is  rod-shaped,  and  stains 
intensely  red  with  Leishman.  It  usually  is  directed  point- 
ing obliquely  towards  the  nucleus.     (Plate  II.,  b). 

These  bodies  are  much  the  same  size  as  blood  platelets, 
but  the  peculiar  chromatin  masses  render  them  easy  to 
recognize.     They  are  frequently  found  in  clumps. 

In  the  peripheral  blood  they  may  be  found  in  thin 
films  within  leucocytes,  but  as  many  leucocytes  as  pos- 
sible should  be  present,  as  only  i  in  ioo  to  500  will 
contain  the  bodies.  The  edges  and  ends  of  the  films 
contain  most  leucocytes,  and  the  number  can  be  in- 
creased by  suddenly  lifting  the  upper  slide  off  the  lower 
one,  in   making  a  film  by  the  ordinary  method.     There 


122 


LEISIIMAX-DOXOYAX    BODIES 


is  always  some  anaemia,  and  degenerate  red  corpuscles 
are  common.  The  leucocytes  are  scanty,  only  2,000  to 
4,000  per  cubic  millimetre.  The  mononuclear  elements 
are  relatively  increased. 

Leishman-Donovan  bodies  were  considered  by  Leishman 
to  resemble,  in  the  arrangement  of  the  chromatin  masses, 
degenerate  forms  of  trvpanosomes.  Laveran  suggested 
a  resemblance  to  piroplasmata. 

Rogers  and  others  have  shown  that  in  cultures  in  sterile 
citric  acid  solution,  i  per  cent.,  to  which  sodium  citrate 
2-5  per  cent,  has  been  added,  the  bodies  become  much 
elongated  and  form  a  flagellum,  showing  that  they  are 
the  resting  stage  of  a  flagellate  (fig.  40ft,  1,  2,  3). 


Fig.  4c. — <z,  Trypanosomas  and  ihe  altered  forms  found  in  culture  ;  />,  Leisli 
man-Donovan  bodies  and  the  altered  forms  found  in  culture. 


It  had  previously  been  shown  that  trypanosomes  could 
be  kept  alive  for  some  time  in  a  medium  rich  in  haemo- 
globin, and  that  forms  (?  degeneration  forms)  were  pro- 
duced which  had  no  flagellum,  whilst  the  form  of  the 
organism  became  round  and  the  centrosome  was  brought 
nearer  to  the  nucleus  (fig.  40^,  1,  2,  3). 

It  is  now  considered  to  be  established  that  the  bodies 
belong  to  the  Mastigophora  or  Flagellata,  but  not  to  the 
genus  Trypanosoma,  as  there  is  no  undulating  membrane 
and  the  flagellum  emerges  from  the  end  at  which  the 
centrosome  is  placed.     It  is  probably  a  Herpetomonas. 

Flagellate  forms  may  be  discharged  from  the  ulcers  in 


DELHI    BOIL  123 

the  intestines  in  kala-azar,  the  disease  due  to  the  Leish- 
man-Donovan bodies. 

Delhi  Boil. — Scrapings  from  the  raw  surface  in  these 
ulcers  show  a  considerable  number  of  bodies  closely 
resembling  Leishman-Donovan  bodies.  The  two  diseases 
are  probably  distinct,  and  therefore  the  parasites  in  Delhi 
boil  are  probably  of  a  different  species  from  those  found 
in  kala-azar. 

The  parasites  of  Delhi  boil  have  been  cultivated  in 
a  medium  similar  to  that  used  for  the  cultivation  of  the 
Leishman-Donovan  bodies.  Exactly  the  same  phases  of 
development  were  observed  as  in  the  parasites  of  kala- 
azar. 

Cryptococcus. — A  parasite,  somewhat  resembling  the 
Leishman-Donovan  body,  has  been  described  under  the 
above  name  in  horses  suffering  from  epizootic  lymphan- 
gitis. The  parasites  are  small  ovoid  or  spherical  bodies 
3  to  5  fi  in  diameter,  found  either  free  in  the  pus  of 
abscesses  or  contained  in  the  large  mononuclear  or 
polymorphonuclear  leucocytes.  They  resemble  the  para- 
sites of  kala-azar  in  man,  but  differ  from  them  in  that  no 
micronucleus  can  be  made  out,  and  the  single  macro- 
nucleus  seems  to  be  less  compact  and  the  individual 
granules  of  the  same  to  be  more  loosely  arranged,  giving 
somewhat  of  a  rosette  appearance.  Some  observers  assert 
that  these  bodies  are  not  protozoa  but  a  variety  of  yeast. 

These  parasites  stain  well  with  Leishman  or  Giemsa's 
stain  and  will  be  found  frequently  in  large  numbers  in 
the  body  of  leucocytes  and  even  invading  the  nucleus. 
(Plate  II.,  a). 


124 


CHAPTER    VII. 

Parasites  other  than  Protozoal  found  in   Human- 
Blood. 

ANIMAL  parasites  belonging  to  higher  orders  of  animal 
life  are  found  in  human  blood. 

Schistosomum  hcematobium  (Bilharzia)  and  S.  japonicum 

frequent  the  veins  of  the  portal  system  and  pelvis  (vide 
Trematoda). 

Nematodes. — One  species  of  filaria  in  the  adult  form 
has  been  once  found  in  the  circulatory  system  of  man 
by  Megalhaes  in  Brazil,  but  no  further  observations  have 
been  made.  The  worms  were  found  in  a  blood-clot  on 
the  left  side  of  the  heart. 

In  the  lower  animals  nematode  worms  are  not  un- 
common in  the  blood.  Filaria  immitis  is  found  in  the 
right  side  of  the  heart  and  pulmonary  vessels  of  the  dog, 
and  in  the  East  and  in  some  of  the  Pacific  Islands  it  is 
exceptional  to  find  a  clog  free  from  these  parasites. 
When  the  worms  are  in  large  numbers  cardiac  dilatation 
and  death  result.  Various  nematode  worms  in  horses 
and  other  animals  cause  "verminous  aneurisms." 

Of  the  human  filaria  the  adults  are  found  in  various 
parts  of  the  body,  whilst  the  embryos  may  be  discharged 
through  an  aperture  in  the  skin,  as  in  F.  medinensis 
(guinea-worm),  and  probably  also  in  F.  volvulus  which  is 
found  in  subcutaneous  cysts  in  patients  in  West  Afrca. 
In  those  filaria  in  which  we  are  at  present  more  specially 
interested  the  embryos  find  their  way  into  the  blood 
and  circulate  with  that  fluid. 

The  filarial  embryos,  or  microfilaria,  as  seen  in  fresh 
blood,  are  clear,  transparent,  worm-like  bodies,  which  are 


MICROFILARIA  1 25 

in  active  movement.  They  are  most  readily  found  in  a 
fresh  fluid  blood  film,  as  the  active  movements  and  the 
disturbance  in  the  red  corpuscles  set  up  by  their  move- 
ment catch  the  eye.  An  inch  or  two-thirds  inch  objec- 
tive is  quite  sufficient  magnification  for  the  detection  of 
the  commonest  micro-filariae,  but  it  is  better  to  use  a  half- 
inch,  as  the  smaller  species  may  be  overlooked  with  the 
two-thirds  objective.  The  film  must  not  be  so  thin  as  that 
required  for  examination  for  malaria  parasites.  No 
special  precautions  are  required,  and  sufficient  blood 
should  be  taken  to  completely  fill  the  space  between  the 
slide  and  cover-glass.  As  the  slide  must  be  kept  for  a 
sufficient  period  to  enable  the  .movements  of  the  worm 
to  cease,  the  cover-glass  should  be  ringed  with  vaseline 
to  prevent  evaporation  of  the  blood. 

To  examine  the  embryos  in  detail,  higher  powers,  in- 
cluding a  one-twelfth  oil  immersion,  are  required.  At 
first  the  movements  of  the  microfilaria  are  so  active  that 
it  is  impossible  to  examine  it  with  these  objectives,  but 
after  some  hours  the  movements  become  much  more 
sluggish,  and  finally  cease.  The  best  time  for  examina- 
tion is  just  before  the  cessation  of  movement  and  the 
death  of  the  embryo.  The  points  to  observe  in  the 
examination  of  the  fresh  embryos  are  : — 

(1)  The  character  of  the  movement  and  whether 
active  locomotion  takes  place  or  whether  the  move- 
ment, however  active,  leads  to  no  progression. 

(2)  The  size  of  the  embryo.  This  is  of  the 
greatest  importance,  as  measurements  of  dried 
specimens  vary  greatly  with  the  rapidity  with  which 
the  film  has  dried. 

(3)  The  shape  of  the  embryo  and  that  of  the  two 
ends. 

(4)  The  presence  or  absence  of  a  loose  sheath. 

(5)  Any  details  of  structure,  and  particularly  the 
presence,  position  and  character  of  any  contractile 
vesicles,  the  so-called  V  spots,  and  the  cephalic 
movements  and  any  appearance  of  armature. 


126 


DIAGNOSIS   OF   MICROFILARIA 


Embryos  can  also  be  readily  observed  in  dried  films. 

The  blood  films  for  diagnostic  purposes  should  be  as 
thick  as  posible.  A  convenient  way  of  making  them  is 
to  allow  three  or  lour  large  drops  of  blood  to  fall  on  a 
slide  close  together  and  smear  them  together  into  a 
space  about  two-thirds  of  an  inch  in  diameter  (fig.  41). 
Allow  to  dry  face  upwards,  protecting  the  films  from 
insects  during  the  process.     Such  a  film  will  be  so  thick 


Fig.  41. 


as  to  be  almost  opaque.  It  must  not  be  fixed.  When 
quite  dry  place  in  water  and  leave  there  till  the  haemo- 
globin is  all  dissolved  out.  It  is  best  to  have  the  film 
side  downwards  in  the  water,  but  not  resting  on  the 
bottom  of  the  vessel.  As  the  haemoglobin  dissolves  out 
it  will  fall  to  the  bottom  of  the  vessel.  It  will  be  found 
better  after  a  few  minutes  to  transfer  the  slide  to  clean 
water,  so  that  it  is  easy  to  observe  when  the  haemoglobin 
is  all  removed. 

Remove  the  slide  from  the  water  and  examine  at  once 
whilst  still  wet.  The  white  corpuscles  will  stand  out 
from  the  film  as  retractile  spots  and  the  white  colourless 
worms  will  also  stand  out  brilliantly. 

If  it    is    preferred  to    stain   the  specimen  it  should  be 


STAINING    OF   MICROFILARIA 


127 


allowed  to  dry  and  fixed  in  alcohol  and  ether.  Any  basic 
stain  gives  good  results,  and  weak  carbol  fuchsin  is  per- 
haps the  best  of  the  aniline  stains.  Haematoxylin  gives 
good  and  permanent  results,  but  the  sheathed  micro- 
filariae do  not  stain  rapidly.  If  the  haemalum  mixture  is 
used  it  should  be  warmed,  and  five  or  ten  minutes  will  be 
required  for  satisfactory  staining  purposes  ;  the  slide 
should  then  be  flushed  and  left  in  water  for  ten  minutes. 
A  good  many  slides  can  be  stained  at  the  same  time. 
For  this  the  staining  vessel  (fig.  42)  is  convenient. 


Fig.  42. 


Counter-staining  brings  out  nothing  more,  but  eosin 
may  be  used  for  this  purpose. 

The  shape  of  the  worm,  and  also  the  sheath  if  present, 
are  well  shown  in  a  specimen  stained  with  hematoxylin. 
The  body  of  the  worm  is  found  to  contain  a  core  of  deeply- 
staining  points  or  nuclei.  These  do  not  extend  to  either 
extremity,  nor  do  they  completely  fill  the  worm,  as  a 
clear,  unstained  portion  is  left  on  each  side.  This  un- 
stained portion  must  not  be  mistaken  for  the  sheath. 
The  sheath  will  be  faintly  stained  and  only  clearly  seen 
at  the  two  ends,  where  it  will  be  found  flattened  on 
itself  and  often  folded  sharply  like  a  piece  of  ribbon. 

In  the  nuclear  core  complete  or  incomplete  gaps  in  the 
mass  of  nuclei  will  be  seen  in  most  micro-filariae.  For 
each   species  the  position  of    these    gaps  is  constant,  or 


128  MICROFILARIA 

nearly  so,  and  consequently  the  exact  position  of  these 
gaps  is  important    for  the  differentiation    and   identifica 

tion  of  species  from  the  examination  of  these  embryos. 

The  arrangement  of  the  nuclear  core  at  the  blunt 
cephalic  end  is  of  particular  importance,  as  it  is  an 
additional  point  used  in  the  diagnosis  of  species.  In 
the  Microfilaria  bancrofti  the  nuclei  are  loosely  arranged 
at  this  vnd,  whilst  in  Microfilaria  loa  they  form  a  compact 
mass  terminating  almost  as  a  straight  line. 

Embryos  of  some  species  of  lilaria  are  not  found  in 
the  same  number  all  through  the  twenty-four  hours. 
During  a  part  of  this  period  they  may  be  found  in 
numbers,  whilst  a  few  hours  later  they  are  found  with 
difficulty  or  not  at  all.  Thus  one  species  has  a  periodicity 
which  is  called  nocturnal,  because  the  embryos  are  found 
in  largest  numbers  in  the  peripheral  blood  at  night ; 
in  other  species  embryos  are  only  found  in  the  daytime 
and  are  said  to  have  a  diurnal  periodicity.  Embryos  of 
other  species  are  found  in  fairly  equal  numbers  at  all 
times  of  the  day  and  night. 

In  any  investigation  of  the  periodicity  of  filarial  em- 
bryos it  is  essential  that  measured  quantities  of  blood 
should  be  examined. 

The  periodicity  can  be  altered  in  the  case  of  Microfilaria 
bancrofti  by  changing  the  habits  of  the  host,  and  cases 
are  fairly  common  in  which  the  periodicity  is  reversed 
without  known  cause.  It  is  still  more  common  to  find 
small  numbers  of  Microfilaria  bancrofti  during  the  day 
and  larger  numbers  at  night. 

The  chief  points  of  difference  in  the  various  embryo 
filariae  are  indicated  in  the  subjoined  table. 

These  points  require  no  detailed  explanation. 

It  is  well  to  draw  the  embryos  accurately  with  a  draw- 
ing camera  or  camera  lucida.  By  substituting  a  scale  for 
the  object,  a  scale  can  also  be  drawn  on  the  same  paper 
and  measurements  made  from  this,  which  is  easier  and 
usually  more  accurate  than  measurements  made  with  a 
micrometer  eyepiece. 


MICROFILARIA — SPKCIKS 


129 


Distance 

Adult 
(known  or 
suspected) 

Name 

Length 

Greatest 

thickness 

Sheath 

Shape  of 
tail 

Periodicity 

of  head 

gap  from 

head 

mm. 

mm. 

mm. 

Microfilaria  noc- 

•317 

•0075 

Present 

Sharply 

Nocturnal 

•052 

F.  bancrofli. 

turia  or  Micro- 

pointed 

in    peri- 

filaria bancrofli 

pheral 
blood 

Microfilaria 

■317 

•007 

Present 

Sharply 

Diurnal  in 

•052 

F.  loa. 

diurna  or  Micro- 

pointed 

periphe- 

filaria loa 

ral  blood 

Microfilaria  Pers- 

•195 

•CO45 

Absent 

Blunt, 

None 

•03 

F.  Persians. 

ians 

truncated 

Mi c  r  of.  1  a  ri  a 

.21 

•005 

Absent 

Sharply 

None 

•03 

F.     demar- 

demarquayi 

pointed 

quayi. 

Mi  c  r  0  fi  I  ar  i  a 

•7.1 

•005 

Absent 

Sharply 

None 

•03 

F.  ozzardi. 

ozzardi 

pointed 

Periodicity  refers  to  the  time  of  appearance  of  embryos 
in  the  peripheral  blood.  With  regard  to  this  periodicity, 
it  was  for  a  long  time  not  definitely  known  what  became 
of  the  embryos  during  the  time  they  were  absent  from  the 
peripheral  blood.  Post-mortem  examinations,  however, 
have  shown  that  in  the  case  of  persons  harbouring 
F.  bancrofti  where  death  has  occurred  during  the  day, 
the  embryos  are  found  in  greatest  numbers  in  the  lungs 
and  large  vessels,  though  some  may  be  found  in  the 
vesssels  of  other  viscera. 

In  sections  of  the  organs  of  such  a  person  the  micro- 
filaria are  found  in  numbers.  The  material  may  be 
imbedded  in  either  celloidin  or  paraffin,  and  should  not 
be  too  thin,  or  such  short  lengths  of  the  microfilarias  will 
be  cut  that  they  cannot  be  easily  identified. 

H hematoxylin  solution,  two  minutes,  is  quite  sufficient 
to  stain  the  embryos  in  section,  and  there  is  no  need 
to  counter-stain.  Transverse  and  oblique  sections  of 
numerous  embryos  will  be  found.  In  places  longer 
lengths,  or  even  complete  embryos,  which  were  lying 
in  the  plane  of  the  section,  may  be  seen. 

As  far  as  is  known,  no  developmental  changes  take  place 
in  the  human  filarial  embryos  in  the  blood  or  human 
tissues,  but  there  is  evidence  that  some  degree  of  growth 
9 


130  FILARIA 

does  take  place  in  some  of  the  avian  microfilaria  whilst 
they  are  circulating  in  the  blood. 

In  the  case  of  the  human  Microfilaria  iioclunia,  the 
next  stage  of  growth  occurs  in  several  species  of  mos- 
quitoes of  different  genera — Culex,  Anopheles,  Mansonia, 
&c. — and  when  a  certain  stage  of  maturity  is  reached  the 
embryos  escape  from  the  proboscis  of  the  mosquito 
and  pass  through  the  skin  into  man.  At  this  stage  the 
embryos  in  the  case  of  F.  bancrofti  are  1*5  mm.  in  length, 
the  alimentary  canal  is  complete,  but  the  sexual  organs 
are  not  developed. 

The  further  development  in  man  has  not  been  traced, 
but  the  adult  forms  of  the  species  F.  bancrofti  have 
been  found  by  many  observers  always  in,  or  in  con- 
nection with,  the  lymphatic  system.  The  other  human 
adult  filariae,  F.  perstans,  F.  demarquayi,  F.  ozzardi,  and 
F.  loa  (the  adult  form  of  Microfilaria  diurna),  are  found 
in  connective  tissue,  either  subcutaneous  or  in  the 
subperitoneal  tissues. 

The  adult  human  filariae  are  not  very  readily  found. 
F.  bancrofti  are  found  in  lymphatics  in  almost  any  part 
of  the  body,  but  as  a  rule,  in  the  cases  of  elephantiasis, 
the  adults  are  long  dead  and  the  positions  they  once 
occupied  only  indicated  by  lymphatic  obstruction.  F. 
perstans,  though  smaller,  are  more  readily  found,  as  they 
occur,  at  any  rate  in  greatest  numbers,  in  subperitoneal 
connective  tissue,  particularly  at  the  base  of  the  mesen- 
tery. F.  demarquayi  has  been  found  by  Dr.  Galgey  in 
the  same  position,  and  F.  ozzardi  has  been  once  found 
in  the  subserous  connective  tissue  of  the  anterior  abdo- 
minal wall. 

Adult  filaria,  when  found,  are  occasionally  in  a  condi- 
tion of  partial  or  complete  calcification.  These  calcified 
filaria  occur  fairly  commonly  in  the  pelvis  of  the  kidney, 
in  lymphatic  glands,  and  occasionally  in  lymphatics  else- 
where in  the  body.  This  should  be  borne  in  mind  when 
a  search  is  being  made  for  the  adults. 

F.  loa  can  be  seen  when  it   passes  under  the  skin   or 


FILARIA  131 

conjunctiva.  It  is  difficult  to  extract,  for  as  soon  as  an 
incision  is  made  in  the  skin  it  rapidly  moves  away. 

F.  immitis,  the  "worm  in  the  heart"  of  dogs,  is  found 
in  the  cavity  of  the  right  side  of  the  heart  and  the  pul- 
monary vessels.  When  only  one  or  two  worms  are  pre- 
sent they  are  usually  in  the  smaller  pulmonary  arteries. 
Avian  filariae  occur  in  many  positions.  Some  species  are 
found  in  loose  connective  tissues,  as  in  the  neck,  others 
in  the  limbs,  and  particularly  in  thickenings  about  the 
claws  ;  others  in  the  submucous  tissues,  as  in  the  crop  ; 
and  others  in  the  blood-vessels,  and  even  in  the  pouches 
formed  by  the  semi-lunar  valves.  Adult  filariae  are  easily 
mistaken  for  empty  blood-vessels,  small  nerves  and  shreds 
of  fibrous  tissue.  They  are  more  readily  recognized  with 
slight  magnification,  and  for  this  purpose  a  watchmaker's 
glass  of  about  five-inch  focal  length  is  very  useful.  Those 
mounted  in  horn  are  best,  and  should  be  perforated  at 
the  sides,  otherwise  moisture  condenses  on  the  lens.  The 
advantage  of  these  glasses  is  that  both  hands  are  free,  and 
it  is  easy  to  learn  the  use  of  this  simple  lens. 

In  searching  tissues  for  filaria  a  dark  surface,  such  as 
a  slab  of  slate,  makes  a  good  background,  and  the  rough 
surface  of  the  slate  prevents  the  specimen  slipping  about. 
The  tissue  should  be  kept  wet  with  normal  saline  solu- 
tion, as  this  keeps  it  transparent.  The  dissection  should 
not  be  niade  with  the  tissues  floating  in  water  or  salt 
solution,  as  strands  of  tissue  are  much  more  readily 
twisted  or  ravelled  out  if  floating,  and  would  be  mistaken 
for  filaria. 

Description  of  Adult  Filaria. — Some  authors  con- 
struct a  formula  for  the  description  of  filariae  based  on  the 
relative  positions  of  various  structures  and  the  measure- 
ment of  the  worm  at  these  places.  The  unit  of  measure- 
ment is  the  one-hundredth  part  of  the  length  of  the  worm, 
so  that  the  measurements  are  percentages  of  the  length. 
Five  measurements  are  taken  by  the  author  of  the  method 
— Cobb — commencing  from  the  head  :  the  base  of  the 
oesophagus,  the  nerve  ring,  the  cardiac  constriction,  the 


I  12 


F1LARIA 


fourth  at  the  vulva  in  the  female  and  the  middle  of  the 
male,  and  the  fifth  at  the  anus  (fig.  43). 

Many  of  these  points  are  very  difficult  to  make  out  in 
the  human  filariae.  In  living  filariae  the  first  of  them  is 
very  variable  in  the  same  individual.  As  the  head  and 
neck  are  capable  of  considerable  contraction,  the  head 
cannot  be  taken  as  a  fixed  point  to  serve  as  the  basis  of 
a  series  of  measurements.  Also  the  whole  formula  is 
based  on  the  assumption  that  the  proportions  of  various 
parts  of  the  body  are  constant  in  different  individuals, 
which,  according  to  Shipley,  is  not  certain. 

Though  we  do  not  consider  that  this  graphic  method 
is  applicable  in  many  cases,  still,  where  possible,  it  may 
be  given. 

The  human  filarial  resemble  each  other  rather  closely 
in  their  adult  forms,  and  some  of  them  require  very 
careful    examination   for    differentiation.       Measurements 


Fig.  43. 


of  the  head  and  tail,  making  due  allowance  for  the  con- 
tractility of  the  worms,  are  of  great  importance.  Par- 
ticular attention  must  be  paid  to  the  transparent  cuticle, 
as  there  are  important  differences  in  its  arrangement  in 
different  species,  and  these  differences  are  constant  for 
the  individuals  of  each  species. 

The  measurements  should  be  made,  where  possible,  in 
the  fresh  worms,  as  serious  shrinking  and  distortion 
occur  with  most  reagents.  Alcohol  and  spirit  cause 
great  distortion.     This  can  be  diminished   by  placing  the 


FILARIA 


133 


Fig.  44. 
Head  of  Filaria  bancrofli,   ? 


Fig.  45. 
Head  of  Filaria  ozzardi,    2 


134 


FILAR  I A 


specimen  first  in  dilute  spirit,  i  to  3  of  water,  for  a  few 
hours,  and  then  gradually  increasing  the  strength,  but 
however  carefully  this  is  done  the  distortion  is  great. 
Much  less  distortion  is  caused  by  spirit  if  the  specimen 
is  first  hardened  in  formalin  2  per  cent. 

A  general  method  for  the  treatment  of  nematodes  is 
as  follows  : — 

(1)  Place  the  worms  alive  in  a  1  per  cent,  saline  solution 
and  shake  up.     This  removes  all  mucus. 


Fig.  46. 
Tail  of  Filaria  bancrofti,   5  . 


Fig.  47 
Tail  of  Filaria  ozzardi,   $ 


(2)  Have  ready  alcohol  (60  to  70  per  cent.)  which  has 
been  heated  to  the  boiling  point.  This  is  best  done  in  a 
porcelain  dish  over  a  flame  protected  by  wire  gauze. 
Transfer  the  worms  from  the  saline  solution  to  the  hot 
alcohol,  dropping  them  in  one  at  a  time,  when  the  worms 
will  die  in  an  extended  position. 

They  may  be  preserved  in  70  per  cent,  alcohol  till 
required  for  examination. 

(3)  For  examination  they  are  placed  in  a  vessel  con- 
taining  a   mixture  of  95  parts  alcohol  and   5  parts  pure 


FILARIA 


135 


glycerine  and  placed  on  a  water  bath.  The  alcohol  is 
thus  evaporated.  In  pure  glycerine  the  worms  become 
transparent.  They  may  be  studied  in  glycerine  or 
mounted  in  glycerine  jelly. 

If  placed  at  once  in  glycerine  without  any  preliminary 
treatment,  there  is,  at  first,  some  swelling,  though  when 


Fig.  48. 

Head  of  Filaria  demarquayi,    J 


Fig.  49. 
Head  of  Filaria perstans,.  ? 


left  long  in  the  glycerine  there  is  a  return  to  a  more 
natural  condition.  The  specimens  so  prepared  are  much 
softened  and  can  very  readily  be  flattened  out,  and  whilst 
thus  gently  compressed  between  two  slides  be  hardened 
in  methylated  spirit  and  finally  in  alcohol,  and  mounted 
after  clearing  in  oil  of  cloves.  Such  specimens  are  very 
transparent  and  do  not  show  much  detail  ;    if,  however, 


F.  banaofli 

/*'.  f-e 

stans 

F.  oz 

zardi 

Female 

Male 

Female 

Male 

Female 

Vale 

Length 

80—90  mm. 

44  mm. 

70—80  mm. 

45  mm. 

81  mm. 



38  mm 

Greatest  breadth 

•23  mm. 

•  1    m  m . 

•  1 2   mm. 

•06  mm. 

■21  mm. 

•19  rum 

Diameter  of  head     ... 

•055  mm. 

■05    mm. 

•07  mm. 

•04  mm. 

■05  mm. 

? 

Character  of  cephalic 
end 

Unarmed. 
Rounded 

Unarmed. 
Rounded 

Unarmed. 
Rounded 

Unarmed. 
Rounded 

Unarmed 
Rounded 

? 

Distance    of     genital 
pore       from       head 
(female) 

"66 — 75  mm. 

— 

-6  mm. 

•71  mm. 

— 

Diameter  at    point   of 
genital  pore 

•14  mm. 

— 

•07  mm. 

— 

•12  mm. 

— 

Distance  from   tail   of 

•225  mm. 

— 

•145  mm. 

— 

•23  mn-. 

— 

anus 

Cuticular      thickening 
on  tip  of  tail 

None 

None 

Double    ter- 
minal cuti- 
cular thick- 
ening 

None... 

None  .. 

Spicules  (male) 

— 

Two  unequal, 
anterior  and 
posterior, 
both   retrac- 
tile 

— 

Two  unequal 
spicules 

— 

? 

Papilla;  (caudal) 

None 

Minute       flat 
papillre  have 
been     de- 
scribed     by 
Leiper 

None 

Four  preanal 
and        one 
postanal. 
Very  close 
to  opening 
of  cloaca 

None... 

None  .. 

Habitat          

Lymphatic  s) 

stem... 

Connective  t 

issue,  usually 

subperitor 

leal 

Geographical       distri- 
bution 

In  most  tropical  regions    ... 

Africa    (West    Coast    and 
Central),  British   Guiana 

British  Guiana  .. 

F.   deviarquayi 


F.  loa 


Female 

65—80  mm. 

•21 — "25  mm. 

"i  —  '09  mm. 

•76  mm. 


•25  mm. 


Cuticular  thick- 
ening over 
tip.  Knobby 
and  irregular 
in  ouiline 


Male 


Not  known 


Subperitoneal  connective  tissue 


West  Indies 


Female 


Male 


50—55  mm.         30— 35  mn, 
'55  mm- 


Rounded  with 
papillae 


2"35  mm. 


•3  mm. 


No  thickening 
over  tip. 
Two  lateral 
alas.  Cuticu- 
lar bosses  not 
found  at  tip, 
but  over  the 
greater  part 
of  the  body 


F.  magalhfiesi 


I  75  mm. 


Thicke  ning 
over  tip.  The 
"bosses"  so 
abundant  over 
the  cuticle  in 
the  body  of 
the  worm  are 
not  found  at 
the  tip 

Two  unequal, 
anterior  and 
posterior 


Three  preanal 
pairs  and  two 
postanal.  The 
last  are  very 
small 


Connective  tissues,  subcuta- 
neous, subconjunctival,  or  in 
the  deeper  parts  of  the  limbs. 

West  Africa 


Female 

155  mm. 

•6 — "7  mm. 

•06  mm. 

2'56mm. 

•58  mm. 
•13  mm. 

None 


Male 

83  mm. 

•3 --4  mm. 

•04  mm. 


None. 


Two  spicules. 


Four  preanal 
and  four 
postanal. 


Left  side  of  heart. 


Brazil. 


n8 


FILARIA 


they  are  slowly  stained  with  very  dilute  solution-,  of 
stains,  such  as  dilute  borax  carmine,  before  placing  in 
glycerine,  many  details  of  structure  are  brought  out  well. 
They  can  also  be  stained  with  well-diluted  hematoxylin, 

and  subsequently  slightly  decolorized  with  dilute  acid 
spirit  £  per  cent,  to  show  eggs  and  embryos  in  situ. 


Fig.  50. 

Tail  of  Filaria  demarqiiayi,    $ 


FIG.  51. 
Taii  of  Filaria  perstans,    2 


Many  filarise  show  fairly  well  when  mounted  direct  in 
glycerine  jelly,  but  these,  after  a  time,  become  distorted. 
If  previously  hardened  by  placing  first  in  i  per  cent. 
formalin  for  two  days  and  then  in  2  per  cent,  formalin 
for  two  days,  and  then  kept  in  5  per  cent,  formalin  for 
some  days,  they  can  be  mounted  in  glycerine  jelly,  or 
even  in  Farrant's  solution,  and  retain  their  natural  size 
and  appearance. 

The  different  points  enumerated  in  the  tabular  form 
can  usually  be  made  out,  but  to  see  either  the  genital  pore 


BACTERIA   IN   BLOOD  1 39 

or  the  anal  opening  that  portion  of  the  worm  must  be 
viewed  in  profile.  It  is  therefore  necessary  to  turn  the 
worm  gently  before  mounting  so  that  they  can  be  seen. 
This  can  generally  be  effected  by  slightly  moving  the 
cover-glass  by  pressure  on  its  edge  with  a  needle  so  as  to 
roll  the  worm  over  slightly. 

The  points  of  difference  and  resemblance  are  shown  in 
the  table  (pp.  136  and  137  and  figs.  44-51)  for  the  known 
adult  human  filariae.  The  description  of  F.  ozzardi  is 
from  a  single  specimen.  The  embryos  or  microfilariae  of 
F .  ozzardi  and  F.  demarquayi  correspond  in  every  respect. 
As  the  single  adult  found  of  F.  ozzardi  differs  from  the 
specimens  of  F.  demarquayi  in  some  respects,  the  worms 
cannot  be  considered  as  the  same,  but  further  specimens 
are  required  before  the  question  of  the  identity  of  the 
species  can   be   considered  to  be  settled. 

Examination  of  the  Blood  for  Pathogenic 
Bacteria. — Most  of  the  organisms  found  in  blood  films 
are  due  to  contamination  with  skin  organisms  during  the 
preparation  of  the  film.  To  avoid  this,  the  finger,  which 
for  this  purpose  is  the  most  convenient  part  to  examine, 
should  be  well  washed  with  2  per  cent,  lysol,  and  then 
wrapped  in  a  1  in  500  sublimate  compress  covered  with 
gutta-percha  tissue  for  twelve  hours.  The  first  drop  of 
blood  should  be  rejected  as  the  most  likely  to  be  con- 
taminated. Thick  and  thin  films  should  be  taken  and 
rapidly  dried.  The  thin  films  can  be  stained  by  Louis 
jenner's  or  Leishman's  stains,  or  the  film  can  be  fixed 
and  stained  by  any  of  the  methods  used  for  bacteria.  In 
thick  films,  after  drying,  the  haemoglobin  can  be  removed 
by  placing  in  sterilized  water,  and  the  film  afterwards 
fixed  and  stained. 

In  most  cases  organisms  are  present  in  such  minute 
numbers  that  they  will  not  be  found  by  this  method,  and 
the  drops  of  blood  obtained  should  be  used  for  making 
cultures.  The  organisms  of  plague,  septicaemia,  tubercle, 
&c,  may  be  found  in  the  blood,  but  great  caution  must 
be  exercised  as  mistakes  are  frequent.     Cultivation  of  the 


[40  BACTERIA    IN    BLOOD 

organisms  from  blood  drawn  from  a  vein  by  a  hypo- 
dermic syringe  is  more  satisfactory.  For  this  purpose 
a  few  drops  of  blood  should  be  placed  in  each  of  a 
series  of  flasks  containing  some  20  cc.  of  broth,  so  that 
great  and  rapid  dilution  of  the  blood  takes  place.  The 
leucocytes  will  fall  to  the  bottom  of  such  flasks,  and  will 
not  destroy  so  many  of  the  organisms  which  may  be 
present  in  the  blood. 


I4I 


CHAPTER   VIII. 

Certain   Properties   of   Blood  Plasma  and  Blood 

Serum. 

The  living  blood  in  the  body  is  composed  of  a  fluid 
element — the  plasma — in  which  are  suspended  the  solid 
elements,  red  and  white  corpuscles,  blood  platelets,  and 
at  times  parasites.  After  death,  and  when  the  blood  is 
shed,  coagulation  occurs,  the  blood  plasma  being  con- 
verted into  a  jelly-like  mass  which  soon  contracts,  and  a 
fluid — the  blood  serum — separates.  Parasites  are  usually 
included  in  the  coagulum  as  well  as  the  more  solid  ele- 
ments of  the  blood,  such  as  the  corpuscles. 

Blood  Plasma. — For  certain  purposes  it  is  desirable 
that  the  blood  should  be  kept  fluid  and  coagulation 
prevented.  This  is  requisite  when  we  wish  to  inject 
blood  containing  living  parasites,  such  as  filarial,  or  try- 
panosomes,  &c,  or  to  obtain  certain  constituents  of  the 
blood,  such  as  the  white  blood  corpuscles.  If  the  blood 
is  allowed  to  coagulate,  parasites  contained  in  it  are 
usually  entangled  in  the  blood-clot.  To  prevent  this, 
the  blood  should  be  quickly  mixed  with  a  10  per  cent, 
solution  of  citrate  of  sodium.  One  part  of  this  solu- 
tion, if  rapidly  and  thoroughly  mixed,  will  prevent  the 
coagulation  of  twenty-five  parts  of  blood.  Twice  the 
quantity  of  a  5  per  cent,  solution  is  somewhat  easier  to 
work  with  and  equally  effective — others  use  a  normal 
citrate  of  soda  solution,  a  much  larger  quantity  being 
required.  In  these  mixtures  the  blood  corpuscles  are 
not  destroyed. 

To  obtain  blood  in  quantity  such  as  is  required  for 
injecting  into  animals,  it  is  best  to  plunge  a  hypodermic 


I42  BLOOD   PLASMA 

needle  into  a  distended  vein.  The  median  basilic  or 
median  cephalic  will  be  found  most  convenient.  An 
all-glass  syringe  should  he  used.  No  powerful  suction 
is  required;  if  the  point  of  the  needle  is  in  the  vein,  by 
holding  the  syringe  horizontally  very  little  suction  is 
necessary.  When  possible  injections  should  be  made  at 
once  with  unmixed  blood  ;  if  this  is  not  possible  the 
blood  should  be  citrated. 

To  study  the  characters  and  special  properties  of  either 
the  red  or  white  corpuscles,  coagulation  must  be  pre- 
vented in  the  same  way  and  the  corpuscles  rapidly 
separated  by  centrifugalizing  the  blood.  Such  corpuscles 
may  then  be  transferred  by  means  of  a  pipette  to  normal 
saline  solution,  and  will  retain  their  properties  for  a 
considerable  time. 

Coagulation  Time. — Blood  varies  greatly  in  the  rapidity 
and  firmness  with  which  it  coagulates,  and  the  time  re- 
quired is  influenced  by  various  diseases.  Methods  of 
estimating  the  coagulation  time  for  clinical  purposes  are 
not  very  satisfactory,  and  all  determinations  must  be 
made  at  a  constant  temperature. 

Wright's  method  is  to  draw  up  blood  into  a  series  of 
capillary  tubes  of  uniform  calibre,  and  attempt,  by  blow- 
ing at  intervals  of  half  a  minute,  to  dislodge  the  blood. 
When  it  cannot  be  dislodged  it  has  coagulated,  and  the 
time  it  has  taken  is  the  coagulation  time.  This  simple 
method  appears  to  give  as  good  results  as  any. 

Specific  Gravity. — The  specific  gravity  of  the  blood  is 
another  valuable  element,  and  is  not  easily  determined 
accurately  with  the  small  amounts  of  blood  that  can  be 
used  for  clinical  purposes. 

Blood  is  dropped  into  a  series  of  fluids  of  known 
specific  gravity  varying  from  1035  to  1068,  and  the 
specific  gravity  of  the  fluid  in  which  the  blood  neither 
sinks  nor  rises  is  of  the  same  specific  gravity  as  the 
blood.  The  fluids  chiefly  used  are  glycerine  and  water 
or  chloroform  and  benzol  in  varying  proportions. 

Chemical  Reaction. — The  reaction  of  the  blood  can  be 


BLOOD  I43 

determined  either  by  using  glazed  litmus  paper  previously 
soaked  in  chloride  of  sodium  solution,  or  a  plaster  of 
Paris  disc  soaked  in  neutral  litmus  solution. 

Spectroscopic  Examination. — -In  addition  to  haemoglobin 
we  may  have  in  the  blood  in  some  cases  derivatives  or 
modifications  of  haemoglobin.  A  small  direct  vision 
spectroscope  is  the  most  satisfactory  method  of  deter- 
mining the  presence  of  these  substances.  The  blood 
should  be  laked  by  the  addition  of  distilled  water  to 
render  it  sufficiently  translucent.  If  it  be  desired  to 
determine  the  presence  or  absence  of  haemoglobin  from 
the  serum  another  specimen  of  the  blood  should  be 
allowed  to  coagulate,  and  when  the  serum  has  separated 
that  should  be  examined  separately.  The  diluted  blood 
should  be  placed  in  a  small  vessel  with  two  plane  sides 
inclined  towards  each  other  at  an  acute  angle,  so  that 
varying  thicknesses  of  the  fluid  can  be  examined. 

Either  ordinary  daylight  or  a  lamp  can  be  used,  and 
the  spectrum  should  first  be  focussed  as  sharply  as 
possible  and  the  slit  closed  as  much  as  is  convenient  to 
bring  out  Fraunhofer's  lines  distinctly. 

The  spectra  of  oxyhemoglobin  and  reduced  haemo- 
globin can  be  readily  obtained  from  the  same  specimen, 
either  by  shaking  up  with  air  to  oxidize  or  reducing  by 
the  addition  of  ammonium  sulphide  (Plate  III.). 

Methaemoglobin  gives  two  additional  lines,  as  seen  in 
the  diagram,  and  the  two  lines  between  the  D  and  E  are 
further  apart  and  faint ;  on  the  addition  of  alkali  the 
spectrum  changes  and  becomes  more  like  that  of  oxy- 
hemoglobin (Plate  III.).  Methaemoglobin  is  of  con- 
siderable importance,  as  it  colours  the  urine  brown  and 
not  red.  If  no  ■  spectroscopic  examination  is  made  it 
will  usually  be  overlooked.  In  some  of  the  mildest  cases 
of  "  Blackwater  Fever"  methaemoglobin  and  not  haemo- 
globin may  be  found.  Urobilin  is  shown  by  the  single 
broad  band  between  E  and  F  (Plate  III.).  Bile  in  human 
urine  causes  no  definite  banding. 

The  coloured  plate  gives  the  spectra    of  haemoglobin 


144  TOXICITY 

and  its  derivatives  ;  some  of  these  are  only  formed  under 
artificial  circumstances  and  consequently  are  of  little 
practical  clinical  value. 

The  colouring  matter  of  blood  is  haemoglobin,  it  forms 
some  90  per  cent,  of  the  red  corpuscles,  and  is  not  found 
in  blood  plasma,  nor,  when  the  blood  coagulates,  in  the 
serum.  It  can,  however,  be  readily  removed  from  the 
red  corpuscles  by  the  addition  of  water  either  to  the 
fluid  blood  or  to  the  freshly  dried  blood. 

Advantage  is  taken  of  this  property  when  thick  films 
are  made,  as  in  examining  for  filaria,  in  order  to  render 
a  thick  Aim  transparent.  To  prevent  the  occurrence  of 
this  solution  in  making  preparations  for  the  examina- 
tion of  thin  films,  "  fixing  agents,"  such  as  alcohol,  per- 
chloride  of  mercury,  formalin  solution  or  vapour,  or 
heat,  are  employed. 

Estimation  of  Tonicity. — Different  specimens  of  blood 
vary  in  the  power  of  the  corpuscles  to  retain  their  haemo- 
globin. Though  distilled  water  will  remove  the  haemo- 
globin completely  from  the  corpuscles  in  fluid  blood, 
saline  solutions  over  a  certain  strength  will  not  remove  it. 
This  resistance  or  "tonicity"  of  the  blood  corpuscles  is 
measured  by  the  strength  of  saline  solution,  which  is  just 
sufficient  to  prevent  the  solution  of  the  haemoglobin. 
Such  a  solution  is  said  to  be  "  isotonic."  Normal  saline 
solution  75  per  cent,  prevents  the  solution  of  haemo- 
globin in  blood.  A  series  of  weaker  solutions  differing 
in  the  amount  by  *02  per  cent,  is  made,  and  a  drop  of 
blood  is  dropped  into  each,  and  after  shaking  allowed 
to  stand. 

The  weakest  salt  solution  that  does  not  cause  solution 
of  the  haemoglobin  is  the  index  of  the •" tonicity "  of  the 
blood  used. 

The  strength  of  that  solution  gives  the  isotonic  strength, 
which  is  the  measure  of  the  resistance  of  the  blood, 
normally  0*46  to  0*48  per  cent.  A  less  accurate  but  more 
convenient  method  is  to  mix  a  measured  amount  of  the 
blood  with  a  measured  amount  of  a  solution,  such  as  a 


Plate  EI. 

BLOOD-SPECTRA    COMPARED    WITH    SPECTRUM    OF 
ARGAND-LAMP. 


1  Spectrum  of  Argand-lamp  with  Fraunhofer  lines  in  position. 

2  Spectrum  of  Oxyhemoglobin  in  diluted   blood. 

3  Spectrum  of  reduced   Haemoglobin. 

4  Spectrum  of  carbonic  oxide   Haemoglobin. 

5  Spectrum  of  acid   Haematin   in  etherial  solution. 

6  Spectrum  of  alkaline  Haematin. 

7  Spectrum  of  Chloroform  extract  of  acidulated  Ox'bile. 

8  Spectrum  of  Methaemoglobin   (alkaline.) 

9  Spectrum  of  Haemochromogen. 

I  0  Spectrum  of   Haematoporphyrin. 

Most    of    the    above    Spectra    have    been    orawn    from    observations   by 
Mr.     W.     LEPRAIK,  F.C.S. 

Fig.  52. 


TONICITY  145 

3  per  cent,  solution,  which  is  well  above  the  isotonic 
strength,  and  add  gradually  measured  amounts  of  water 
till  the  solution  of  the  haemoglobin  takes  place  ;  from  this 
the  strength  of  the  fluid  which  just  causes  solution  can 
be  calculated. 

Wright's  tubes  with  the  air  and  mixing  chambers  are 
convenient  for  the  purpose.  The  tonicity  of  the  blood  is 
of  considerable  importance,  as  a  decrease  in  the  tonicity 
often  precedes  a  haemolytic  attack.  This  occurs  in  black- 
water  fever,  and  persons  whose  blood  is  of  a  low  degree 
of  tonicity  should  not  be  allowed  to  live  in  countries 
where  this  disease  is  endemic. 

These  methods  assume  that  the  red  corpuscles  in  the 
blood  are  all  equal  as  regards  their  tonicity,  or  nearly  so. 
This  is  not  the  case,  as  even  in  healthy  blood  an  occa- 
sional corpuscle  will  be  found,  that  will  be  decolorized 
in  a  stronger  solution  of  salt  than  the  others,  and  also  a 
few  will  retain  their  haemoglobin  when  all  the  others  are 
decolorized.  With  healthy  blood  the  great  majority  of 
the  corpuscles  may  be  equal,  but  with  blood  in  some 
diseases,  a  much  larger  number  of  red  corpuscles  are  of  a 
lower  degree  of  tonicity  than  the  average. 

To  determine  the  range  of  variation  of  tonicity  in  the 
corpuscles  the  blood  should  be  first  well  diluted  with  a 
strong  salt  solution,  4  per  cent.,  which  is  hypertonic  to 
all  corpuscles.  The  mixing  can  be  done  in  one  of 
Wright's  tubes.  The  tube  is  allowed  to  stand  and  the 
corpuscles  will  fall  to  the  bottom,  and  by  heating  the 
air  chamber  can  then  be  expelled  into  a  clean  watch 
glass. 

Hanging  drop  preparations  of  this  blood,  diluted  with 
one,  three,  or  seven  parts  of  distilled  water,  will  be  equiva- 
lent to  2  per  cent.,  1  per  cent,  and  '5  per  cent,  of  salt  solu- 
tion. An  examination  of  these  hanging  drops  will  show 
if  any  considerable  proportion  of  the  corpuscles  have  lost 
their  haemoglobin.  If  none  or  very  few  are  decolorized 
with  the  1  per  cent,  salt  solution  the  remainder  of  the  red 
corpuscles  in  the  watch  glass  in  4  per  cent,  salt  solution 
10 


146  TONICITY 

should  be  diluted  with  three  parts  of  distilled  water  in 
one  of  Wright's  tubes  and  well  mixed  in  the  mixing 
chamber.     The  fluid  can  be  expelled  into  a  clean  wat  ch 

glass  and  a  series  of  dilutions,  as  hanging  drops,  made. 
One  part  diluted  with  one  of  water  will  give  -5  per  cent., 
with  two  of  water  '33  per  cent.  Two  parts  of  the  diluted 
blood  with  one  of  water  will  give  '66  per  cent.,  and  so 
on.  In  this  manner,  by  examining  a  series  of  these 
hanging  drops,  and  determining  the  proportion  of  the 
"shadow"  corpuscles  which  can  be  easily  seen  with  an 
oil  immersion  lens  if  the  light  is  cut  off,  the  proportion  of 
corpuscles  of  lower  index  of  tonicity  than  represented  by 
these  solutions  can  be  determined. 

When  the  haemoglobin  is  dissolved  in  the  serum  the 
blood  is  said  to  be  "  laked."  Dissolved  haemoglobin  is 
found  in  the  serum  in  acute  haemolytic  processes,  but 
appears  to  be  rapidly  removed  either  by  the  hepatic  or 
renal  cells,  converted  by  the  liver  into  bilirubin,  or 
deposited  in  the  subcutaneous  tissues.  The  yellow  tint 
of  skin  and  conjunctiva  in  some  diseases  which  simulate 
jaundice  is  of  this  nature,  and  is  called  haematogenous 
jaundice.  The  yellow  tinge  round  old  bruises  is  due 
to  the  solution  of  the  haemoglobin  of  the  extravasated 
blood. 

Normal  blood  serum  is  ''hypertonic,"  that  is,  not  only 
is  it  sufficient  to  prevent  the  solution  of  haemoglobin 
from  the  red  corpuscle,  or  is  isotonic,  but  is  considerablv 
above  that  strength.  This  excess  of  tonic  value  is  not 
simply  due  to  the  amount  of  salts  ;  it  varies  consider- 
ably and  is  estimated  by  determining  the  dilution  with 
water  required  to  render  it  isotonic  as  regards  normal 
red  corpuscles.  This  can  be  determined  either  by  using 
a  series  of  dilutions  and  dropping  (with  distilled  water, 
or  as  a  series  of  hanging  drops  in  Wright's  tubes)  blood 
into  each,  or  by  diluting  to  a  known  extent  a  mixture  of 
blood  and  serum. 

Blood  Scrum. — Blood  serum  is  required  for  several 
purposes,  notably  for  the  demonstration  of  the  presence 
or  absence  of  specific  agglutinins. 


SERUM  147 

The  glass  tubes  devised  by  Wright  afford  a  ready 
method  of  obtaining  and  diluting  serum.  The  simplest 
form  is  to  draw  out  a  piece  of  glass  tubing  of  about  one- 
quarter  inch  diameter.  In  drawing  out  the  tube  it  is  well 
to  rotate  it  in  the  flame  until  it  is  quite  soft  at  the  required 
place,  then  removing  it  from  the  flame,  pull  steadily.  In 
this  way  tubes  of  more  uniform  size  are  obtained  than  if  the 
traction  be  exercised  while  the  tube  is  still  in  the  flame. 
The  thin  tube  thus  formed  should  be  broken,  and  at  a 
convenient  distance  another  portion  of  the  tube  should 
be  heated  and  pulled  out  in  the  same  manner,  or  bent  as 
shown  in  fig.  53.*    One  of  the  capillary  extremities  should 


Fig.  53. 

be  sealed.  A  puncture  with  a  broad  needle  or  small 
knife  should  be  made  in  the  skin  and  the  upper  half  of 
the  unexpanded  tube,  that  towards  the  sealed  end  should 
be  heated  in  the  flame  of  a  spirit  lamp,  which  is  lighted 
and  placed  close  at  hand  before  the  skin  is  punctured. 
Holding  the  lower  part  of  the  tube  which  has  not 
been  drawn  between  the  fingers  to  make  sure  that  it  is 
not  too  hot,  the  open  drawn-out  end  is  placed  in  the 
exuding  blood.  As  the  air  in  the  tube  cools  and  con- 
tracts the  blood  will  be  drawn  up  into  the  tube.  If  there 
is  not  enough  or  the  blood  is  not  drawn  entirely  up  into 
the  thick  part  of  the  tube  by  the  time  it  is  cool,  the  sealed 

*  We  are  indebted  to  the  kindness  of  the  proprietors  of  the  Lancet 
for  the  use  of  fig.  53. 


148  SERUM 

end  can  he  broken  off  and  the  upper  end  of  the  thick 
tubing  again  heated  and  the  same  end  again  sealed. 
The  contraction  of  the  air  will  be  sufficient  to  draw  more 
blood  up,  and  the  blood  already  in  the  tube  higher  up. 
When  sufficient  blood  is  in  the  tube  and  the  tube  is  cool, 
the  lower  end  through  which  the  blood  entered  can  be 
scaled.  The  tube  is  now  placed  on  its  side  horizontally 
till  the  blood  coagulates,  and  is  then  placed  vertically,  so 
that  as  the  serum  is  expressed  by  the  contraction  of  the 
clot  it  will  run  down  into  the  narrow  part  of  the  tube. 
In  this  way  clear  serum,  free  from  blood  corpuscles,  can 
be  obtained  without  using  a  centrifuge.  Capillary  vac- 
cination tubes  can  be  used  to  collect  the  blood,  but  will 
require  to  be  centrifugalized  to  obtain  clear  serum. 


Fig.  54. 


Serum,  however  prepared,  requires  dilution  for  most 
purposes,  and  the  degree  of  dilution  is  important.  The 
graduated  pipette  of  a  h?emocytometer  may  be  used  for 
this  purpose.  A  very  convenient  method  of  obtaining 
any  degree  of  dilution  is  by  Wright's  tubes.  A  piece  of 
glass  tubing  is  drawn  out  sharply  in  the  middle  so  as  to 
make  a  short,  sharp  constriction.  About  an  inch  and  a 
half  from  this  constriction  on  each  side  the  tubing  is 
drawn  out  into  a  long,  thin  capillary  tube.  One  of  these 
is  broken  off  and  sealed,  and  the  other,  preferably  the 
more  uniform  and  thicker,  is  touched  with  a  file,  broken 
off  square  and  left  open.  The  part  above  the  middle 
constriction  with  the  sealed  capillary  tube  attached  is 
called  the  air  chamber,  and  that  below  in  connection  with 
the  open  capillary  tube  is  called  the  mixing  chamber. 
A  narrow  mark  is  made  on  the  open  capillary  tube  with 
a  grease  pencil  about  an  inch  or  less  from  the  open  end. 
This  distance  depends  on  the  calibre  of  the  tube,  the 
greater  this  is  the  shorter  the  distance  from  the  open  end 


DILUTION    OF   SERUM  149 

to  the  mark,  as  the  volume  of  the  column  of  fluid  between 
the  open  end  and  the  mark  is  the  unit  of  measurement. 
The  finger  or  ear  is  pricked,  and  from  the  blood  obtained 
the  serum  is  allowed  to  separate  as  above  and  blown  out 
into  a  sterile  watch  glass.  The  air  chamber  is  then 
well  heated,  and  the  open  end  of  the  tube  is  placed 
in  the  blood  serum  till  the  serum  runs  up  to  the  mark. 
The  tube  is  then  removed  from  the  serum,  and  a  little 
air  enters  the  tube  as  the  air  in  the  hot  air  chamber 
contracts,  the  open  end  is  then  placed  in  the  diluting 
fluid  and  withdrawn  as  soon  as  the  fluid  reaches  the  grease 
pencil  mark ;  it  is  then  withdrawn,  but  as  soon  as  air 
has  entered  the  tube,  it  can  be  replaced  in  the  fluid  and 
again  withdrawn  when  the  mark  is  reached.  This  can 
be  repeated  as  long  as  the  air  in  the  air  chamber  contracts. 
If  repeated  nine  times,  there  will  be  nine  parts  of  the 
diluent  to  one  of  the  blood  serum.  As  the  fluid  by  con- 
traction of  the  air  in  the  air  chamber  is  all  drawn  up  into 
the  mixing  chamber,  it  can  there  be  well  mixed  by  rapidly 
rotating  between  the  palms  of  the  hands. 

This  procedure  would  give  a  dilution  of  1  in  10,  and 
by  continuing  the  process,  greater  dilutions  would  be 
obtained;  but  it  is  better,  if  high  dilutions  are  required, 
such  as  1  in  100  or  1  in  1,000,  after  well  mixing  the  serum 
and  diluent  to  expel  a  part  of  it  into  a  sterilized  watch 
glass  by  heating  the  air  chamber. 

The  expelled  diluted  serum  is  further  diluted  in  a 
second  tube  in  exactly  the  same  manner  as  the  first 
dilution,  and  from  this  a  third,  and  in  turn  a  fourth, 
dilution  can  be  made.  The  tubes  with  the  diluted  serum 
may  be  sealed  up  and  kept  for  some  time  if  necessary. 

If  it  be  desired,  a  known  amount  of  a  broth  culture  of 
an  organism  can  be  in  the  same  manner  drawn  up  into 
the  mixing  chamber  and  there  mixed  with  diluted  or 
undiluted  serum. 

Wright  uses  india-rubber  teats  to  draw  up  the  fluid 
(vide  fig.  55),  but  as  in  the  Tropics  india-rubber  does  not 
keep  well,  the  air  chamber  drawn  out  into  a  long  capillary 


15°  SERUM 

tube  is  more  satisfactory.  Wright,  when  using  an  air 
chamber,  blows  it  out  into  a  bulb  so  as  to  have  a  larger 
volume  of  air,  but  a  smaller  air  chamber  is  sufficient  in 
most  cases,  and  if  it  cools  too  rapidly,  so  that  the  air 
ceases  to  contract,  the  sealed  end  may  be  broken  off,  and 
whilst  the  tube  is  still  open,  the  air  chamber  can  be  heated 
and  the  tube  again  quickly  sealed.  This  can  be  repeated 
as  often  as  one  wishes  if  a  large  volume  of  serum  or 
blood  is  required  in  the  mixing  chamber. 


Fig.   55- 

Instead  of  serum,  the  blood  itself  can  be  mixed  with  a 
diluent  in  a  similar  manner,  and  the  diluted  blood  used 
for  counting  leucocytes  or  red  corpuscles.  It  is  necessary 
that  the  diluent  should  be  one  that  will  prevent  coagula- 
tion and  will  not  cause  destruction  of  the  red  corpuscles. 
Gower's  solution  is  fairly  satisfactory,  or  if  it  be  desired 
to  stain  the  leucocytes,  Toisson's  fluid  may  be  used.  The 
mixing  for  uniform  and  successful  results  must  be  done 
quickly,  otherwise  part  of  the  blood  may  coagulate  or 
the  corpuscles  adhere  together  in  masses. 

In  addition  to  agglutinins  other  substances  may  be 
formed  in  serum  as  a  result  of  infection  by  micro- 
organisms. These  include  the  toxins  and  antitoxins, 
i.e.,  the  poisonous  products  of  the  growth  of  organisms 
and  substances  that  neutralize  these  poisonous  products  ; 
also  hemolysins  or  substances  that  have  a  destructive 
action  on  blood  corpuscles. 

Precipitins. — A  class  of  substances  which  promise  to  be 
of  much  practical  importance  are  the  precipitins.  It  is 
found  that  if  blood  of  one  animal,  as,  for  instance,  man, 
be  repeatedly  injected  into  a  rabbit,  the  constitutional 
disturbance  set  up  by  the  injections  becomes  less  and 
less,  and  after  a  few  injections  they   cease   to   cause  any 


PRECIPITINS  I5I 

disturbance.  It  is  further  found  that  the  blood  serum 
of  this  rabbit,  immunized  as  to  human  blood,  will  give 
a  precipitate  when  added  to  a  solution  of  human  serum 
or  of  closely  related  animals,  such  as  the  ape,  when  much 
diluted,  but  not  with  solutions  of  serum  of  other  animals, 
such  as  the  Rodentia. 

Similarly,  if  a  rabbit  be  immunized  by  repeated  injec- 
tions of  the  blood  of  any  animal  of  a  different  genus, 
horse,  rat,  pig,  &c,  the  serum  of  the  rabbit  will  give  a 
precipitate  with  solutions  of  the  serum  of  the  horse,  rat, 
and  pig  respectively,  or  animals  closely  related  to  them. 

This  gives  a  new  means  of  grouping  animals  and  pro- 
mises to  be  of  practical  and  medico-legal  value.  The 
immunized  or  test  sera  can  only  be  made  where  proper 
appliances  are  available,  but  appear  to  keep  well. 

The  application  of  the  test  is  easy.  Clean  white  filter 
paper  is  soaked  in  the  fresh  uncoagulated  blood  of  the 
animal  and  allowed  to  dry  in  the  air.  A  portion  of  this 
paper  is  treated  with  normal  saline  solution.  The  clear 
solution  thus  obtained  is  placed  in  a  small  test  tube 
(Durham's  tubes  are  suitable)  and  a  few  drops  of  the  test 
serum  are  added.  A  precipitate  indicates  that  the  blood 
was  either  that  of  the  animal  against  whose  blood  the 
rabbit  was  immunized  or  a  closely  related  one. 

Nuttall  has  made  observations  on  a  large  series  of 
animals,  and  the  results  obtained  have  been  consistent, 
and  in  many  ways  have  thrown  light  on  the  relationship 
of  different  animals.  The  filter  papers,  soaked  in  blood 
and  well  dried  in  the  air,  keep  well  and  give  the 
reaction  after  many  months.  It  is  important  that  blood 
should  be  obtained  from  any  rare  animal  and  examined 
in  this  way  to  aid  in  its  classification.  The  filter  paper 
or  white  blotting  paper  should  be  soaked  in  the  blood 
and  clots  removed.  The  blood  must  be  fresh.  The 
paper  should  then  be  allowed  to  thoroughly  dry,  and,  if 
possible,  wrapped  in  wax  paper  and  sent  to  England 
to  be  tested  with  the  prepared   sera. 

Culture  Medium. — Blood  serum  makes  an  excellent  cul- 


152  OPSONINS 

hire  medium  for  many  organisms,  and  sonic  will  not 
grow,  or  only  grow  with  difficulty,  in  more  artificial 
media. 

For  the  cultivation  of  trypanosomes  the  blood  scrum 
obtained  after  the  blood  has  been  "laked"  is  the  most 
satisfactory  medium.  Such  a  fluid  is  a  solution  of  haemo- 
globin in  diluted  blood  serum. 

Opsonins. — Another  property  of  blood  serum  is  the 
power  it  possesses  of  acting  on  pathogenic  micro- 
organisms, and  so  altering  them  that  they  can  be  taken 
up  and  destroyed  by  the  leucocytes.  The  substances  to 
which  the  blood  serum  owes  this  property  have  been 
named  by  their  discoverer,  Wright,  opsonins  (Latin  opsono, 
I  prepare  food  for).  Wright  believes  that  the  leucocyte 
is  a  constant  factor  in  the  phenomenon  of  phagocytosis, 
and  that  the  variable  and  most  important  factor  is  the 
amount  of  opsonin  in  the  blood  serum.  It  is  further  con- 
sidered probable  that  these  opsonins  have  a  high  degree 
of  specificity,  that  is  to  say,  that  there  is  a  corresponding 
opsonin  for  each  organism  ;  that  for  the  tubercle  bacillus, 
for  example,  has  no  action  on  staphylococci,  and  vice 
versa. 

In  order  to  estimate  the  amount  of  opsonin  present  in 
a  blood  serum  for  a  given  organism  there  is  required  : — 

(1)  The  serum  to  be  tested. 

(2)  The  serum  of  a  healthy  person  to  be  used  as  a 
control. 

(3)  An  emulsion  of  the  given  organism  in  normal  saline 
solution.  This  emulsion  may  contain  either  living  or 
dead  organisms,  and  they  should  be  present  in  such 
numbers  as  to  admit  of  their  being  readily  counted  in 
the  leucocytes. 

(4)  Leucocytes  washed  free  from  plasma. 

To  obtain  these  allow  about  1  c.c.  of  blood  from  a 
healthy  person  to  drop  into  10  c.c.  of  normal  saline 
solution  containing  i£  per  cent,  of  sodium  citrate.  This 
mixture  is  now  centrifugalized  until  the  corpuscles  have 
fallen    to    the    bottom.       The    clear    supernatant    fluid    is 


OPSONINS  153 

then  pipetted  off  and  replaced  by  normal  saline  solution 
and  the  whole  shaken  up.  After  further  centrifugaliza- 
tion  the  supernatant  fluid  is  again  removed,  and  the 
leucocytes,  which  form  a  creamy  layer  on  top  of  the  red 
blood  cells,  are  ready  for  use. 

(5)  Two  pipettes  prepared  as  described  on  p.  150  (fig.  55). 

As  described  under  serum  dilutions,  draw  up  equal 
parts  of  (1)  emulsion  of  organisms  ;  (2)  emulsion  of  leuco- 
cytes ;  (3)  serum  to  be  tested  in  the  order  named.  Expel 
these  on  to  a  glass  slide  and  mix  thoroughly.  Afterwards 
draw  the  mixture  into  the  pipette  and  seal  off  the  tip. 
Remove  the  rubber  teat  and  place  the  pipette  in  an 
incubator,  or  in  a  vessel  of  water  at  370  C,  carefully 
noting  the  exact  time.  Now  prepare  a  second  pipette  in 
the  same  way,  using  this  time  the  normal  serum  instead 
of  the  serum  to  be  tested.  Place  this  under  precisely  the 
same  temperature  conditions. 

At  the  end  of  fifteen  minutes  in  the  case  of  each  pipette 
expel  the  contents  on  to  a  slide,  mix  well,  and  prepare 
smears.  The  smears  are  then  fixed  and  stained  by  a 
method  suitable  for  the  demonstration  of  the  organisms. 

The  stained  smear  should  then  be  examined  with  a 
lens  of  low  powTer,  and  a  part  selected  where  leucocytes 
are  present  in  greatest  numbers. 

On  examination  with  the  oil  immersion  lens,  it  will 
be  found  that  organisms  have  been  taken  up  by  the  poly- 
morphonuclear leucocytes.  The  number  of  organisms 
in  eighty  such  leucocytes  should  be  counted,  in  the  one 
case  where  the  serum  to  be  tested  was  used,  and  also  in 
the  other  case  where  the  control  serum  was  used. 

The  ratio  between  these  two  figures  gives  the  opsonic 
index.  For  example,  if  in  the  smear  prepared  from  the 
mixture  in  which  the  serum  to  be  tested  was  used,  it  was 
found  that  80  polymorphonuclear  leucocytes  contained 
95  organisms,  and  in  the  other  smear  the  same  number 
of  polymorphonuclear  leucocytes  contained  190  organisms, 
then  the  opsonic  index  of  the  serum  under  examination 
for  that  organism  is  jrfb  =  "5. 


*54 


CHAPTER    IX. 
Arthropoda — Insecta. 

With  the  progress  of  the  enquiry  into  the  causation 
of  disease  it  has  come  to  be  of  importance  that  the 
investigator  of  tropical  diseases  should  possess  some 
knowledge  of  the  blood-sucking  flies,  mosquitoes,  ticks, 
fleas,  &c,  which  are  known  or  suspected  to  be  con- 
cerned in  the  transmission  of  various  infections.  A  brief 
account  is  here  given  of  the  more  important  members  of 
the  zoological  division  Arthropoda,  to  which  these  carriers 
of  disease  belong. 

The  Arthropoda  are  bilaterally  symmetrical  segmented 
animals  with  a  thick  chitinous  cuticle.  To  some  of  the 
segments  are  attached  articulated  ambulatory  limbs — it 
is  from  this  feature  that  the  name  of  the  phylum  or  sub- 
kingdom  was  derived. 

The  segments  vary  in  number,  and  in  some  arthropods 
the  segmentation  is  not  obvious  externally,  but  is  inferred 
from  the  arrangement  of  internal  structures,  such  as 
nerve  ganglia.  The  cephalic  segments,  three  in  number, 
are  always  fused  into  one  mass,  the  head — the  cephalic 
appendages  are  modified  for  purposes  of  mastication  or 
suction  and  sensation.  The  three  or  four  segments 
behind  the  head  are  often  fused  more  or  less  completely 
to  form  the  thorax,  which  may  be  united  to  the  head  to 
form  the  cephalo-thorax.  To  the  thorax  are  attached 
the  legs.  The  abdominal  segments  are  usually  distinct, 
but  may  be  fused  and  united  to  the  cephalo-thorax,  as  in 
some  of  the  Arachnida.  The  abdominal  appendages  are 
essentiallv  those    connected    with    excretion    and    repro- 


ARTHROPODA  1 55 

duction,  but  may  be  so  modified  as  to  form  "stings,"  or 
weapons  of  offence  and  defence. 

The  sub-kingdom  or  phylum  Arthropoda  is  divided 
into  four  groups  : — 

(i)  Myriapoda  (centipedes,  millipedes,  &c). 

(2)  Iusccici  (horse-flies,  butterflies,  mosquitoes,  &c). 

(3)  Arachnida  (spiders,  ticks,  &c). 

(4)  Crustacea  (cyclops,  &c). 

The  Insecta  and  Arachnida  include  the  most  important 
carriers  of  disease,  and  will  be  considered  first. 

Class  Insecta  or  Hexapoda. — The  term  Insecta  was 
originally  employed  to  embrace  all  those  animals  whose 
body  is  externally  divided  into  segments,  including  butter- 
flies, beetles,  centipedes,  scorpions,  &c.  It  is  now  used  in 
a  much  more  restricted  sense  to  apply  to  such  arthro- 
pods as  have  six  walking-legs.  The  Insecta  have  the 
body  distinctly  divided  into  three  regions — head,  thorax 
and  abdomen.  They  take  in  air  by  means  of  tracheae, 
a  system  of  tubes  ramifying  throughout  the  body  and 
opening  externally  by  means  of  orifices  placed  in  pairs 
at  the  sides  of  the  body.  The  appendages  forming 
the  mouth-parts  are  paired,  and  consist  of  mandibles, 
maxillae  and  labium,  the  pair  in  this  latter  part  being 
combined  to  form  a  single  body.  To  the  head  are  also 
attached  a  pair  of  antennas. 

There  are  two  pairs  of  wings,  but  one  or  both  of  these 
may  be  rudimentary  or  modified  for  purposes  other  than 
flight  :  sometimes  even  rudimentary  wings  appear  to  be 
absent.  The  wings  are  always  placed  on  the  thorax,  and 
to  this  region  also  are  attached  the  three  pairs  of  legs. 

Insects  in  many  of  the  orders  undergo  a  variety  of 
changes  of  form  in  the  course  of  their  development. 

In  the  system  here  adopted  the  insects  are  divided  into 
eleven  great  groups  or  Orders,  the  characteristics  of 
which  we  give  in  brief.  By  "  mandibulate  mouth  "  is 
meant  one  in  which  the  mandibles  or  maxillae,  or  both, 
are  fitted  for  biting,  crushing  or  grasping  food.  The 
term  "suctorial"  implies  that  some  of  the  mouth-parts 


156  INSECTA 

arc  modified  to  form  a  tubular  suctorial  apparatus;  this 
is  frequently  protected  by  a  modification  of  other  parts, 
which  act  as  a  sheath. 

(1)  Aptera. — Wingless  insects.  All  wingless  insects  do 
not,  however,  belong  to  this  order.  Mouth  mandibulate, 
or  very  imperfectly  suctorial. 

Two  sub-orders. — (i.)   Thysanura  /  (ii.)  Collembola. 

(2)  Orthoptera. — Four  wings,  the  front  pair  being 
leather-like,  and  usually  smaller  than  the  hind  pair,  which 
are  membranous  and  contract  after  the  manner  of  a  fan. 
Mouth  mandibulate. 

This  order  includes  earwigs,  cockroaches,  grasshoppers, 
crickets,  &c. 

(3)  Neuroptera. — Two  pairs  of  membranous  wings, 
frequently  with  much  network  ;  the  front  pair  similar 
to  the  hind  ;  the  latter  with  little  or  no  fanlike  action  in 
closing.     Mouth  mandibulate. 

This  order  includes  dragon-flies,  may-flies,  termites,  &c. 
There  are  some  parasitic  wingless  forms,  such  as  "  bird- 
lice."  Adult  dragon-flies  are  destructive  to  other  insects, 
and  their  larvae  are  very  destructive  to  aquatic  larvae, 
such  as  those  of  mosquitoes.  Attention  to  the  breeding 
of  some  species  of  dragon-flies,  particularly  of  the 
Agrionidce,  is  important,  as  these  breed  in  places  similar 
to  those  in  which  larvae  of  Culicidae  are  usually  found. 

The  larvae  of  Agrionidce  have  short  rounded  bodies. 
Their  mouth-parts  are  modified  to  form  a  protrusible 
mask  with  which  they  seize  their  prey.  These  larvae 
lurk  at  the  bottoms  of  pools,  puddles  and  streams,  and 
are  the  most  important  of  the  natural  enemies  of  mos- 
quito larvae.  The  adult  Agrionidce  can  be  distinguished 
by  the  peculiar  position  of  the  wings,  which  are  always 
inclined  backwards,  never  at  right  angles  to  the  body  as 
in  other  dragon-flies. 

(4)  Hymenoptera. — Two  pairs  of  membranous  wings, 
linked  together  with  little  hooks,  the  front  pair  being 
larger  than  the  hind,  which  are  always  small,  and  do  not 
fold  up  in  repose.  Mouth  mandibulate,  sometimes  also 
provided  with  a  tubular  proboscis. 


INSECTA  157 

This  order  includes  bees,  wasps,  ants,  &c. 

(5)  Coleoptera. — Two  pairs  of  wings,  the  front  pair 
shell-like  and  forming  cases  which  meet  together  over 
the  back,  so  as  to  lose  entirely  the  appearance  of  wings 
and  to  conceal  the  delicate  membranous  hind  pair. 
Mouth  mandibulate. 

This  order  includes  the  beetles. 

(6)  Lepidoptera. — Two  pairs  of  large  wings  covered 
with  scales.     Mouth  suctorial. 

This  order  includes  butterflies  and  moths. 

(7)  Diptera. — One  pair  of  membranous  wings.  Mouth 
suctorial,  but  varying  greatly,  sometimes  penetrating  as 
well  as  suctorial. 

(8)  Thysanoptera. — Two  pairs  of  very  narrow  fringed 
wings.     Mouth  imperfectly  suctorial. 

This  order  includes  thrips. 

(9)  Hemiptera  or  Rhyncota. — Two  pairs  of  wings,  the 
front  pair  either  leather-like,  with  membranous  apex,  or 
entirely  parchment-like  or  membranous.  Mouth  perfectly 
suctorial,  and  modified  into  a  definite  beak,  which  is  bent 
so  as  to  be  flattened  out  under  the  head  and  thorax. 

This  order  includes  bed-bugs,  scale-insects,  cicada,  &c. 

(10)  Siphonaptera. — Wings  absent.  Mouth  suctorial  and 
piercing.  Thorax  composed  of  three  separate  parts  not 
fused  together.  Legs  powerful  and  adapted  for  jumping. 
This  order  comprises  fleas. 

(11)  Aiioplcura. — Wings  absent.  Mouth  suctorial  and 
of  peculiar  construction,  of  a  totally  different  nature  to 
the  mouth  parts  of  other  orders.  This  order  includes 
pediculi. 

Metamorphosis. — Conspicuous  changes  after  birth,  or 
metamorphosis,  is  one  of  the  most  striking  phenomena 
of  insect  life. 

The  more  highly  specialized  insects,  such  as  Lepido- 
ptera, Diptera,  Hymenoptera,  Coleoptera,  and  most 
Neuroptera,  undergo  profound  changes  of  form  known  as 
indirect  or  complete  metamorphosis.  For  example,  the 
egg  of  a  mosquito  or  house-fly  produces  a  larva.     The 


158  INSECTA 

larva  feeds  and  grows,  casting  its  skin  from  time  to  tunc, 
but  not  changing  markedly  in  form  till  it  becomes  a 
pupa.  From  this  pupa,  in  which  internal  development 
only  takes  place  and  no  food  is  taken,  the  imago  or  adult 
sexual  form  emerges.  Thus  these  insects,  at  different 
stages  in  their  life-history,  assume  very  dissimilar  forms. 

In  other  less  highly  specialized  insects,  such  as 
Orthoptera  and  Hemiptera,  the  imago  or  adult  resembles 
in  external  form  the  young  at  birth,  except  that  the  latter 
is  devoid  of  wings  and  mature  sexual  organs.  This  is 
known  as  direct  or  incomplete  metamorphosis.  As  there 
is  no  distinction  between  larva  and  pupa,  it  is  usual 
to  employ  the  term  "  nymph "  to  describe  the  stage 
between  egg  and  imago  in  such  insects. 

In  cases  of  complete  metamorphosis,  it  is  usually 
possible  to  determine  the  order  to  which  a  larva  belongs, 
though  the  actual  form  of  the  larvae  and  adults  is  so 
dissimilar.  Most  dipterous  larvae  have  no  legs  ;  they 
may  be  aquatic,  as  in  the  case  of  mosquitoes,  or  head- 
less maggots,  as  in  those  of  the  Muscidae.  Coleopterous 
larvae  have  three  pairs  of  legs,  whether  they  are  like 
maggots,  worms,  or  wingless  insects.  Lepidopterous 
larvae  have  not  only  three  pairs  of  legs,  but  also  supple- 
mentary legs  or  pseudopodia  on  the  abdominal  segments  ; 
these  differ  in  appearance  from  the  true  legs  and  are  not 
jointed. 


'59 


CHAPTER    X. 

DiPTERA. 

Many  members  of  the  order  Diptera  are  known  to  be 
concerned  in  the  causation  of  diseases  of  man  and  of 
animals. 

Diptera  may  be  harmful  to  man  and  animals  in  a 
variety  of  ways,  by  biting,  by  living  as  parasites,  especially 
in  ;the  larval  stage,  either  internally  or  externally,  and 
by  carrying  disease  germs  either  as  direct  agents  or  as 
intermediate  hosts  for  parasites. 

(i)  Certain  species  are  noted  for  their  virulent  bite  ; 
such  insects  apparently  secrete  a  poisonous  or  irritating 
saliva.  Whether  the  virulence  of  flies'  saliva  varies  at 
different  times  is  not  known ;  the  dissimilar  effects  pro- 
duced by  particular  species  upon  man  at  different  periods 
and  upon  different  individuals  may  be  due  to  varying 
susceptibility.  There  is  no  doubt,  however,  that  at 
certain  times  biting  insects  are  more  venomous  than  at 
others. 

Diptera  feed  both  by  night  and  by  day ;  as  a  rule  each 
species,  often  each  family,  has  its  particular  feeding  time. 
The  gadflies  (Tabanidce),  for  instance,  only  feed  during 
the  day  ;  Cnlicina  usually  feed  at  night,  but  some  species 
are  day-feeders,  and  some  may  feed  either  by  day  or  by 
night;  Auophdina  chiefly  but  not  exclusively  by  night. 
Fleas,  or  Pulicidcv,  are  almost  exclusively  nocturnal. 

(2)  Internal  Parasitism  is  fairly  common  in  this  order 
of  insects,  and  man  may  be  the  host.  Human  dipterous 
parasites  are  nearly  always  found  as  such  in  their  larval 
state.     There  are  some  notable  exceptions  in  which  the 


160  DIPTERA 

adult  is  the  parasite,  as  the  Jigger  Flea  (Sarcopsylla 
penetrans).     The  Larvae  of  diptera  parasitic  in  man  and 

in  animals  produce  what  is  technically  called  Myiasis. 
These  parasitic  larvae  may  be  situated  internally  (internal 
myiasis)  or  externally,  or  under  the  skin  (cutaneous 
myiasis).  Grubs  such  as  the  horse-bots,  or  larvae  of  Gas- 
trophilus  equi,  may  live  and  develop  in  the  stomach  and 
intestines  of  the  horse,  the  horse  forming  a  definite  host. 
Grubs  may  exist  in  the  intestines  of  man,  as  the  Antho- 
myia  larva.',  by  chance  occurrence  and  not  normally. 
There  are  no  known  dipterous  larvae  which  live  and 
develop  only  in  man's  intestines.  Cases  of  internal 
myiasis  in  animals  are  common,  in  man  rare. 

Cutaneous  myiasis  is  much  more  common  in  man. 
The  eggs  of  various  diptera  are  deposited  on  sores  and 
wounds,  and  the  grubs  feed  in  such  places  (Lucilia  and 
Calliphora),  or  the  larvae  may  live  under  the  skin  (Dcr- 
matobia),  or  even  penetrate  the  organs  of  sight.  External 
myiasis  refers  not  only  to  the  skin  but  includes  cases  of 
insect  invasion  of  the  external  openings  of  the  body, 
such  as  the  nose,  orbits,  ears,  vagina,  rectum,  &c. 
(Screw-worm,  Compsomyia  macellaria) . 

Cases  of  internal  myiasis  require  the  most  careful  in- 
vestigation, as  diptera  may  deposit  not  only  eggs  but 
living  young  on  faeces  directly  they  are  voided,  and  these 
maggots  may  be  thought  to  have  been  passed  per  anum. 
There  are,  however,  well-authenticated  cases  of  internal 
myiasis,  even  in  England. 

(3)  Diptera  often  feed  indiscriminately  upon  man  and 
animals.  In  this  way  a  biting  fly  may  carry  germs  of 
some  disease  from  animal  to  man,  such,  for  instance,  as 
anthrax,  or  from  man  himself  to  a  fellow-creature. 
Another  source  of  infection  of  disease  in  man  in  which 
diptera  play  a  prominent  part  is  not  due  to  biting  diptera 
alone,  but  to  germs  being  carried  from  faecal  matter  in 
latrines,  &c,  by  all  kinds  of  carrion  and  foul-feeding 
flies,  to  man's  food  and  drink  (typhoid  fever,  &c). 

An    important    role   is  played    by  diptera    as  definitive 


DIPTERA  161 

hosts  of  human  parasites,  such  as  the  malaria  parasites, 
and  as  intermediate  hosts  for  the  Filarice. 

Order  DlPTERA. — Flics  with  the  anterior  pair  of  wings 
membranous,  except  in  the  case  of  certain  parasitic  forms, 
as  the  sheep  "  tick,"  which  are  wingless.  The  posterior 
pair  of  wings  is  transformed  into  a  pair  of  club-shaped 
processes,  the  halteres  or  balancers.  The  head,  thorax 
and  abdomen  are  distinct.  The  head  is  very  variable  in 
shape.  There  are  usually  two  large  compound  eyes,  and 
ocelli  may  be  present.  The  antennae  are  very  variable 
and  present  important  characters  which  are  of  importance 
in  classification ,  Certain  families  have  antennae  which 
consist  of  a  number  of  segments  approximately  similar 
to  one  another  and  arranged  in  a  linear  manner  (fig.  56, 
1  and  2).  The  number  of  the  segments  in  this  division 
varies  in  the  different  families,  from  eight  to  sixteen. 
Diptera  having  this  form  of  antennae  are  called  Nemocera 
or  Nematocera.  The  majority  of  insects  popularly  known 
as  "  flies  "  have  antennas  of  another  form,  namely,  three 
segments,  the  third  of  which  is  of  different  form  according 
to  genus  or  species,  and  bears  on  its  front  a  tine  projecting 
bristle  frequently  feathered,  the  arista  (fig.  56,  6).  Between 
the  two  forms  of  antennae  described  there  exists  a  variety 
of  intermediate  forms.  In  these  latter  there  are  one  to 
three  segments,  and  a  terminal  appendage,  which  is  fre- 
quently annulated  (fig.  56,  5,  Tabanidce),  or  maybe  hairlike 
(fig.  56,  3,  Asilidcc).  Flies  with  these  forms  of  antennae  are 
called  Brachycerous.  Exceptional  forms  of  antennae  are 
found  in  the  parasitic  flies  of  the  series  Pupipara. 

The  mouth  is  suctorial,  and  in  some  the  parts  are 
adapted  for  piercing.     The  normal  mouth-parts  are  : — 

(1)  The  labrum  or  upper  lip. 

(2)  The  mandibles. 

(3)  The  maxillae. 

(4)  The  labium  or  lower  lip. 

(5)  The  hypopharynx. 

Jointed  appendages,  the  maxillary  palpi,  are  also  present ; 
the  labial  palps  are  represented  by  the  labellae,  which  are 
11 


l62 


AN  i  i:\x.k 


jointed  on  to  the  distal  end  of  the  labium.  The  form 
and  sometimes  the  function  of  each  part  varies  in  ea<  h 
group.  The  labium  in  many  species  is  more  or  less 
fleshy  and  acts  as   a  sheathing  organ  ;    the   labrum   and 


^t^7 


Fig.   56. — Antennre. 


i,Culicid;   2,  Simulid  ;  3,   Asilida: ;  4,  Hrematopota  ; 
5,  Tabanid  ;  6,  Muscid. 


hypopharynx    are    often    much    elongated,    and    together 
may  form  a  more  or  less  perfect  tube. 

The  space  between  the  eyes  is  called  the  vertex,  that 
part  in  front  of  the  eyes  the  frons,  the  part  behind  the 
occiput  ;  the  sides  the  gence,  or  cheeks  ;  and  the  mouth 
parts  arise  from  a  projection  in  front,  the  clypeus. 


MOUTH    PARTS 


163 


Many  diptera  have  a  peculiar  structure  in  the  form  of 
a  vesicle  on  the  head  called  a  "fttilinum."  This  is  a 
bladder-like  expansion  in  front  of  the  head,  which  appears 
as  the  fly  emerges  from  the  pupa.  It  serves  to  rupture 
the  hard  shell  in  which  the  fly  is  enclosed.  This  ptilinum 
becomes  completely  inverted  in  the  mature  fly,  being 
represented  externally  by  a  space,  the  "  lunula"  under 
an  arched  suture,  extended  over  the  point  of  insertion 
of  the  antennae. 


FlG.  57. — Mouth  of  an  Empis.  a,  Lower  lip  or  labium;  b,  labella ;  ct 
stylets  or  maxillee;  d,  hypopharynx  ;  f,  upper  lip  or  labrum  ;  g,  maxillary 
palp.     (After  Meinert.) 


The  head  is  joined  to  the  thorax  by  a  narrow  neck 
at  the  back  of  the  head  called  the  nape.  The  thorax  may 
have  all  three  segments  distinct,  or  the  pro-  and  meso- 
thorax  may  fuse ;  the  former  is  usually  small,  the  latter 
large  ;  the  metathorax,  more  commonly  known  as  the 
metanotum,  is  small.  The  prothorax  is  most  pronounced 
in  the  N  ematocera  and  forms  either  two  prothoracic  lobes 
or  a  narrow  collar  ;  a  portion  of  the  mesothorax  is  cut 
off  behind  by  a  depressed  line,  forming  the  scutellum  ; 
a  transverse  suture  may  sometimes  be  seen  on  the  meso- 
thorax running  across  from  the  base  of  the  wings,  and 
there  is  also  a  prominent  groove  above  the  root  of  the 
wings,  along  which  there  are  often  characteristic  bristles. 

The  pleura  or  sides  are  built  up  of  several  pieces,  and 
lie  below  the  meso-  and  metanotum. 

The  wings  have  a  variable  number  of  veins,  which  are 


164  WINGS 

both  longitudinal  and  transverse.     The  figure  given  here 
is  of  a  Daddy-long-legs  {Tipula). 

In  the  centre  will  be  seen  a  space  surrounded  by  veins 
— the  discal  cell  (fig.  58,  9).  On  the  fourth  longitudinal 
vein  that  bounds  this  cell  in  front  will  be  seen  a  short 
connecting  vein — the  anterior  cross- vein  ;  this  always 
connects  the  fourth  longitudinal  vein  behind  with  the 
third  in  front,  and  the  cell  behind  is  always  the  discal 
cell  (9)  ;  between  the  second  and  third  longitudinal  veins 
are  the  marginal  cells.  The  other  cells  are  shown  in  the 
figure. 


Fig.  58. — Wing  of  Tipula.  a,  Costal  vein  ;  b,  mediastinal  vein  ;  c,  first 
longitudinal  vein  ;  d,  second  longitudinal  vein ;  e,  third  longitudinal  vein  ; 
/,  fourth  longitudinal  vein  ;  g,  fifth  longitudinal  vein  ;  h,  sixth  longitudinal 
vein  ;  i,  seventh  longitudinal  vein,  i  and  2,  Mediastinal  cells ;  3  and  4, 
sub-marginal  cells  ;  5,  anterior  basal ;  6,  posterior  basal  ;  7,  anal ;  8,  posterior 
marginal;  9,  discal  cell.     (After  Loew.) 


The  longitudinal  veins  are  known  as  follows  : — 

The  costal  (a)  ; 

Auxiliary,  mediastinal,  or  subcostal  (b) ; 

First  longitudinal  vein  (c) ; 

Second  longitudinal  vein  or  radial  (d)  / 

Third  longitudinal  vein  or  cubital  {e)  ; 

Fourth  longitudinal  vein  or  discoidal  (/)  / 

Fifth  longitudinal  vein  or  postical  (g) ; 

Sixth  longitudinal  vein  or  anal  (h)  ; 

Seventh  longitudinal  vein  or  axillar  rib  (/'). 
On  the  hind  margin  of  the  wing  near  the    base  there 
is  often  a  more   or  less  free  lobe,  the  "  alula,"  and  still 
nearer  the  base  or  placed  on  the  sides  of  the  body  two 


HALTERES  165 

other  lobes,  the  one  nearer  the  alula,  called  the  "  anti- 
squama,"  the  other  the  "  squama,"  which  covers  the 
haltere. 

The  halteres  or  balancers  may  be  hidden  by  the 
squamae,  as  in  certain  Muscidce.  In  such  cases  the  fly 
is  said  to  be  calyptrate. 

The  legs  are  attached  to  pro-,  meso-  and  meta-thorax  ; 
they  usually  terminate  in  ungues  or  hooks,  and  pnlvilli  at 
the  base  of  the  ungues,  in  the  form  of  two  pad-like  fleshy 
cushions,  but  the  latter  may  be  absent.  In  certain  flies 
we  find  between  them  the  empodium,  a  median  appendage 
in  the  form  of  a  pad,  bristle,  or  spine. 


Fig.  59. — Ease  of  wing,  calyptrate  diptera.     c,  Haltere  hidden  by 
(a)  squama. 

The  abdomen  is  composed  of  nine  segments,  but  they 
are  not  as  a  rule  all  shown.  The  male  genitalia  or  hypo- 
pygium  is  of  importance  for  differentiation  of  genera 
and  species. 

All  parts  of  the  body  may  bear  bristles  (chcetce)  which 
are  important  in  classification  (chcetotaxy) . 

The  larvae  of  all  diptera  have  neither  true  nor  false  legs, 
but  may  or  may  not  have  a  distinct  head. 

The  pupae  may  be  either  naked  or  enclosed  in  the 
hardened  larval  skin  or  puparium  (fig.  60). 

The  production  of  living  young  occurs  in  some  groups- 
In  the  forest-flies  and  sheep-ticks,  or  "  keds,"  the  young 


j  66 


CLASSIFICATION 


may  be  born  as  fully  matured  larvae  in  a  puparium  case, 
which  is  at  first  white,  but  soon  darkens.  In  the  Glossina 
the  larva  is  passed  fully  mature,  and  travels  into  suitable 
ground,  and  there  becomes  a  pupa. 


FlG.  60. — Puparium  of  a  "  Screw-worm  "  (enlarged). 


Classification  of  Diptera. 

A  satisfactory  classification  of  Diptera  is  not  yet  agreed 
upon  by  entomologists.  At  one  time  it  was  suggested 
that  the  order  should  be  divided  into  two  great  groups, 
Nemocera  and  Brachycera,  according  to  the  structure 
of  the  antennas,  as  described  above.  Later  Brauer  pro- 
posed a  classification  based  mainly  on  development  as 
shown  by  larval  and  pupal  characters,  into  two  sub- 
orders, Orthorrhapha  (straight-seamed)  and  Cyclorrhapha 
(circular-seamed).  The  characters  of  these  groups  were 
defined  as  follows  : — 

Sub-order  i.  ORTHORRHAPHA. — Larva  with  a  distinct 
head ;  pupa  either  free  or  encased  in  the  larval  skin 
(puparium)  ;  the  pupal  skin  always  bursts,  for  the  exit  of 
the  imago,  in  a  T-shaped  opening  on  the  back  of  the 
anterior  end,  rarely  in  a  transverse  slit  between  the  eighth 


CLASSIFICATION    OF   DIPTERA  167 

and  ninth  segments.     Imago  without  the  frontal  lunula 
and  ptilinum. 

Sub-order  2.  CYCLORRHAPHA. — Larva  without  any 
distinct  head  ;  pupa  always  in  a  puparium  (fig.  60  and 
fig.  79,  a)  ;  imago  always  escapes  via  a  more  or  less 
circular  opening  at  the  anterior  end  (fig.  79,  c).  Frontal 
lunula  always  present  in  the  mature  fly,  as  there  is  a 
ptilinum  when  it  first  emerges. 

Sub-order  I.  Orthorrhapha. — The  pupa  escapes  from  the  larval 
skin  either  through  an  anterior  T-shaped  opening,  or  (rarely) 
through  a  posterior  transverse  slit :  adults  without  a  frontal 
lunule : — 

1.  Nematocera.     Flies  with  four-  or  five-jointed  palpi  and  many- 

jointed  antennae,  the  segments  of  which,  except  the  basal  two, 
are  similar  and  are  often  fringed  with  long  hairs  : — 

i.  Nematocera  vera.  Antennae  long  and  frequently  with  whorls 
of  hairs  :  legs  long  and  slender  :  abdomen  usually  long  and 
slender.  Examples:  Craneflies,  Midges,  Gnats,  Mosquitoes, 
Chironomidae. 

ii.  Nematocera  anomala.  Antennae  of  many  small  segments,  but 
short  and  without  whorls  of  hairs  :  abdomen  usually  stoutish  : 
legs  shorter  and  stouter  than  in  Nematocera  vera.  Examples  : 
March-flies,  Buffalo-gnats.     Simulidae. 

2.  Brachycera.     Flies  with  one-  or  two-jointed  palpi,  and  usually 

short  three-jointed  (sometimes  four-  or  five-jointed)  antennae  : — 
iii.  Brachycera  anomala.     Third  segment  of  antennae  ringed  as 
if  composed  of  several  small  segments  fused  together  :  body 
without    strong  bristles.      Examples :    Horse-flies,  Soldier- 
flies.     Tabanidas; 
iv.  Brachycera  vera.      Third   segment    of  antennae  not  ringed, 
but  usually  bearing  a  bristle  or  style  (antennae  sometimes 
four-  or  five-jointed) :  usually  with  strong  bristles.    Examples : 
Snipe-flies,  Robber-flies,  Dance-flies. 
Sub-order  II.     Cyclorrhapha. — The  pupa  escapes  from  the  larval 
skin  through  an  anterior  circular  opening  :  adults  with  a  frontal 
lunule  :  antennae  short,  usually  three-jointed,  the  third  segment 
with  a  bristle  or  style  : — 

1.  ASCHIZA.     Flies  without  a  frontal  suture.      Examples:   Syrphus 

flies,  Big-eyed  flies. 

2.  Schizophora.      Flies  with  a  frontal  suture.      Examples  :  Bot- 

flies, Muscids,  Glossina,  Stomoxys. 
Sub-order  III.     Pupipara. — Larva  nourished  within  the  parent  and 
not   born   till  it    is    ready  to   change  into  a  pupa.     Examples : 
Tick-flies,  Hippoboscidae,  Bat-ticks,  Bee-louse. 


l68  ORTHORRHAPHA   NEMOCERA 

ORTHORRHAPHA    NEMOCERA. 
NEMOCERA    VERA. 

Family  Cecidomyid.-k  (Gall  Midges). — Small,  slender 
flies  with  long  antennae,  with  bead-like  segments;  pro- 
boscis short,  elongated  in  one  genus  only.  Abdomen 
composed  of  eight  segments.  Wings  usually  hairy  ;  no 
alula  ;  never  more  than  five  longitudinal  veins,  usually 
only  three,  the  first,  third  and  fifth ;  fourth  and  sixth 
may    be    present.       Costal    vein    encloses    entire    wing  ; 


Fig.   6i. — Wing  of  a  Cecidomyia. 

fifth  vein  forked  ;  only  one  basal  cell.  Larvae  vegetable 
feeders  ;  most  produce  galls.  A  few  live  as  parasites  in 
society  of  plant  lice.  Larvae  with  fourteen  segments  and 
possess  an  "anchor  process  "  under  the  head  end  of  body. 
The  proboscis  is  elongated  in  the  genus  Clinorrhyncha 
(Loew),  and  directed  downwards.  They  are  often  injuri- 
ous to  crops,  but  only  exceptionally  cause  annoyance  to 
man  by  biting. 

Family  Culicid^e  (Mosquitoes). — Proboscis  elongated 
for  piercing.  Eyes  reniform  ;  ocelli  wanting.  Antennae 
usually  plumose  in  the  male  (except  Sabethes,  Wyeomyia, 
&c).  Thorax  with  large  mesothorax,  narrow  scutellum, 
rounded  metanotum.  Abdomen  composed  of  eight  seg- 
ments. Wings  (figs.  62  and  63)  with  six  longitudinal 
veins,  exclusive  of  the  sub-costal,  and  two  fork-cells ; 
veins  clothed  with  scales  ;  costal  vein  continued  round 
the  border  of  the  wing,  fringed  with  scales.  Head,  thorax 
and  abdomen  usually  but  not  always  scaly.  Palpi  short 
or  long  in  the  female  and  male.  The  females  are 
bloodsuckers    in    many  species.      The  larvae    and    pupae 


NEMOCERA   VERA  169 

are  aquatic.      This    family  is   dealt  with   in  more   detail 
in  a  subsequent  chapter. 

Family  Blepharocerjd,e.  —  These  little  flies  have 
broad  wings  and  long  legs.  The  proboscis  is  elongated, 
and  the  females  in  some  species  {Curupira)  are  blood- 
suckers. The  thorax  has  a  distinct  transverse  suture. 
The  hind  legs  are  longer  than  the  front  ones  and  there 


FlG.  62. — Wing  of  Anopheles  maculipennis. 


Fig.  63.  —  Wing  of  a  Cnlex. 

are  no  pulvilli.  The  broad  wings  are  quite  bare,  there 
is  no  discal  cell,  they  are  iridescent,  and  have  a  secondary 
set  of  fine  network  of  veins.  They  perform  aerial  dances 
like  midges,  especially  near  the  spray  of  waterfalls.  The 
larvae  live  in  rapidly  running  water  fixed  to  stones  by 
suckers.  Some  forms  of  larvae  {Curupira)  are  composed 
of  only  six  or  seven  segments,  with  widely  projecting 
side  lobes  and  small  tracheal  gills  near  the  suckers.  The 
pupae  are  flattened,  inactive,  and  enclosed  in  a  semi-oval 
shell,  the  anterior  end  having  horny  erect  breathing 
tubes  and  suckers  on  the  ventral  surface. 

Family  Chironomid^e  (Midges). — This  family  includes 
the  majority  of  midges  which  are  frequently  taken  for 
Ciilicidce  or  mosquitoes.  They  are  all  small,  delicate, 
gnat-like  flies,  with  small  head,  partly  concealed  by  the 
cowl-like  thorax.  The  antennae  in  the  female  are  thread- 
like and  composed  of  from  six  to  fifteen  segments  ;  in 
the  male  they  are  densely  plumose.  Ocelli  wanting  or 
rudimentary.  Proboscis  short.  The  oval  thorax  has 
no  transverse  suture,  is  bare,  and  projects  more  or 
less  over  the  head.     The  long,  narrow  abdomen  is  com- 


170 


CHIROXOMID.E 


posed  of  segments  and  is  often  semi-transparent  and 
pilose.  The  legs  are  slender  and  rather  long  and  not 
spinose,  but  very  hairy  in  some  tropical  species.  The 
wings  (fig.  64)  are  narrow,  long,  and  bare  or  hairy,  never 
scaly;  the  anterior  veins  darker  than  the  rest;  the  sub- 
costal vein  complete  but  small ;  second  longitudinal  vein 
small  or  wanting  ;  third  longitudinal  vein  sometimes 
forked  close  to  its  origin,  the  upper  branch  often  rectan- 


FlG.  64. — Wing  of  Chironomus 


FlG.  65. — A  Ceratopogon. 

gular ;  fifth  long  vein  forked,  sometimes  the  fourth  ;   the 
costal  vein  always  ends  near  the  tip  of  the  wing. 

Great  numbers  of  this  family  occur  in  all  parts  of  the 
world.  The  members  of  one  European  genus  {Cerato- 
pogon)  (fig.  65)  bite  severely.  They  often  occur  in  swarms, 
dancing  in  the  air.  When  at  rest  they  wave  their  forelegs 
in  the  air.  The  dorsum  of  the  thorax  is  not  produced 
over  the  head  ;  the  palpi  are  four-jointed  ;  the  wings  are 
usually  spotted  (figs.  65   and  66).     Ceratopogon  occur  in 


PSYCHODIDA:  171 

most  countries.  They  are  known  as  "punkies"  or 
"  no-see-um,"  and  cause  great  annoyance  by  their  bites. 
Many  tropical  genera  bite  severely,  and  on  account  of  this 
and  their  small  size  are  frequently  misnamed  "  sand-flies." 


Fig.  66. — Wing  of  Ceralopogoti.     (After  Leonardi.) 

The  larvae  of  Chironomidce  are  mainly  aquatic  and 
worm-like,  often  red  in  colour,  and  the  pupae  are  active, 
and  the  respiratory  tubes  frequently  plumose  ;  they  also 
live  in  damp  earth  and  in  decaying  vegetation,  and  in 
the  Tropics  in  stumps  of  bamboos,  pitcher  plants  and 
other  open  accumulations  of  water  in  plants.  Those  of 
Ceratopogon  and  allied  genera  live  in  the  sap  of  trees,  under 
fallen  leaves,  and  in  decaying  vegetation,  or  are  aquatic, 
and  are  long,  slender,  delicate,  whitish  creatures. 

Family  Psychodid^e  (Owl-midges).  —  Small,  densely 
hairy,  thick-set  insects.  Proboscis  usually  short,  but  in 
one  European  genus  (Phlebotomus)  it  is  long  and  horny  ; 
palpi  hairy  and  composed  of  four  segments.  The  short 
abdomen  is  composed  of  six  to  eight  segments,  hairy. 
The  legs  are  often  short  and  densely  hairy  and  the  claws 
small.  The  wings  are  broad  and  usually  pointed  at  the 
tip,  and  when  at  rest  lie  roof-shaped  over  the  body  ;  they 
are  densely  covered  with  long  hairs  and  are  fringed  with 
hairs;  neuration  mostly  composed  of  longitudinal  veins ; 
the  costal  vein  completely  encloses  the  wing  ;  the  first 
longitudinal  vein  near  the  costa,  the  second  arises  near 
the  origin  of  the  first  and  is  usually  twice  forked,  third 
vein  simple,  fourth  forked,  fifth,  sixth  and  seventh  usually 
distinct,  the  latter  sometimes  wanting.  These  small  flies 
can  at  once  be  told  by  their  moth-like  appearance.  They 
run  well,  but  their  flight  is  weak.  Owl-midges  are  found 
frequently  on  windows  and  in  out-buildings,  especially  in 


172 


NEMOCERA   AXOMALA 


privies.  Tlie  genus  Phlebotomus  and  some  tropical 
genera  bite  severely.  The  larvae  live  in  stagnant  water 
and  decaying  vegetation.  They  are  cylindrical  and  have 
a  short  terminal  breathing  tube.  The  inactive  pupae  have 
two  long  tubular  stigmata. 


Fig.  67. — Phlebotonma,  sp.     (From  Giles's  "  Gnats  or  Mosquitoes. 


NEMOCERA   ANOMALA. 

Family  SlMULlDiE  (Sand-flies).  Usually  called  sand- 
flies, black  flies,  brulots,  buffalo  and  turkey  gnats,  and 
sometimes  mosquitoes.  All  small,  with  oval  thorax 
devoid  of  any  suture.  Cylindrical  abdomen  composed 
of  seven  or  eight  segments.  The  eyes  are  holoptic,  i.e., 
the  two  eyes  meet  in  the  middle  line  in  the  male,  and 
there  are  no  ocelli.  The  male  is  darker  and  more  velvety 
than  the  female.  The  short  antennas  are  composed  of 
ten  or  eleven  bead-like  segments,  the  two  basal  ones 
distinct,  the  rest  closely  united  and  having  no  whorls  of 
hairs  at  the  joints  of  the  segments.  Palpi  composed  of 
four  segments,  the  basal  joint  short,  the  next  two  equal, 
the  last  longer  and  narrowed.  The  legs  short,  thick  ; 
femora  broad  and  flat.  Wings  (fig.  68)  large  and  broad, 
the  anterior  veins  thickened,  remainder  delicate,  costal 
vein  terminates  near  tip  of  the  wing  ;  the  sub-costal 
terminates  in  the  costa  about  half  the  length  of  the  wing  ; 
first  and  third  longitudinal  veins  lie  close  together  ;  fourth 
vein  forked  nearly  opposite  the  anterior  cross-vein  ;  forks 


SIMULIDiE 


173 


terminate  near  the  tip  of  the  wing-  Proboscis  short  with 
strong  horny  lamellae,  consists  of  two  resisting  bristles 
for  puncturing,  and  on  its  sides  two  four-jointed  maxillary 
palps.  These  small  flies  bite  very  severely  and  cause 
much  annoyance.  They  especially  attack  the  eyes, 
nostrils  and  ears  of  both  animals  and  man.  Sand-flies 
occur  in  all  climates.  The  larvae  are  all  aquatic  and 
some  live  in  rapidly  flowing  water  ;  they  attach  them- 
selves to  stones,  plants,  &c,  and  form  elongated  cocoons, 


Fig.  68. — a,  Wing  of  Simulium  ;  b,  Hinder  end  of  Simulium  larva  ; 
c,  fixative  sucker. 


open  above.  They  are  soft  skinned,  with  thickened  ends, 
a  cylindrical  head,  and  on  the  first  segment  a  prominence 
with  bristly  hooks.  The  end  of  the  abdomen  is  provided 
with  appendages,  by  which  the  larvae  attach  themselves 
(fig.  68,  b).  The  pupae  have  the  anterior  end  of  the  body 
free,  and  from  it  pass  out  a  number  of  thread-like  breath- 
ing tubes.  The  flies  are  accused  of  propagating  anthrax  and 
septic  diseases.  Their  punctures  give  rise  to  severe  inflam- 
mation, which  sometimes  results  in  depilation  in  animals. 


'74 


BRACHYCERA 


Orthorrhapha   Brachycera  (Antennae  short). 
Brachycera  axomala. 

Family  Tabanid.e  (Gad-flies). — This  family  includes 
a  number  of  genera,  the  popular  names  being  gad-, 
breeze-  or  horse-flies,  brimps  and  sneggs.  They  are 
mostly   large  and  stout ;    the  head   (fig.  69)   large  ;  eyes 


Fig.  69.— Head  of  Tabanus. 

very  large,  contiguous  in  the  male,  the  upper  facets 
larger  than  the  lower,  usually  with  green  and  violet 
markings  when  alive.  The  antennae  composed  of  three 
segments,  third  segment  composed  of  six  to  eight 
rings ;     no     stylet.       The     proboscis     prominent,    often 


Fig.  70. — Wing  of  a  Tabanus. 

greatly  elongated,  fleshy,  with  pointed  horny  processes ; 
the  female  with  six,  the  male  with  four  stylets ;  the 
former  only  suck  blood.  Palpi  two-jointed,  the  second 
joint  large.  The  abdomen  is  broad,  often  flattened, 
never  slender,  composed  of  seven  segments  (vide  fig.  71). 
The  legs  are  rather  thick,  mid-tibiae  always  with  spurs  ; 
tarsi  with  three  membranous  pads  at  the  tip.  There  are 
never  any  bristles.     The  third  longitudinal   vein  forked. 


TABANID^E  175 

Two  submarginal  and  five  posterior  cells  present ;  anal 
cell  closed  at  or  near  margin  of  wing.     Tegulae  large. 

Mostly  large  flies  which  occur  during  hot  weather  and 
have  remarkable  powers  of  flight.  The  bite  of  the  female 
is  often  severe.  The  eggs  are  spindle-shaped  and  dark, 
and  are  laid  on  leaves  and  stems  of  plants,  and  on  water 
plants.  The  larvae  are  carnivorous  and  feed  upon  snails, 
insect  larvae  and  also  roots;  elongated,  composed  of 
eleven  segments,  jointed,  often  with  retractile  fleshy 
protuberances  ;  the  last  segment  has  a  breathing  pore, 
or  the  last  two  segments  may  form  a  breathing  tube. 
The  pupae  are  free,  and  live  in  earth  and  water. 

The  worst  biting  species  are  found  in  the  following 
genera  :  Pangonia,  Chrysops,  Lepidoselaga,  Hcematopota, 
Therioplectes,  Atylotus  and  Tabanus. 

There  are  two  sections,  distinguished  as  follows  : — 

Hind  tibia?  with  spurs  at  the  tip  Pangonince . 

Hind  tibiae  without  spurs  Tabanince. 

The  following  characters  separate  the  above-mentioned 

genera : — 

Pangonince. 

Third  joint  of  antennae  eight  ringed,  the  first  ring 
slightly  the  longer  ;  the  fourth  posterior  cell 
open  ;  proboscis  often  very  long     Pangonia. 

Third  joint  composed  of  five  rings,  the  first  of  which 
is  much  longer  than  the  following  ;  the  second 
joint  of  antennae  as  long  as  the  first ;  wings  with 
dark  areas;  three  ocelli;  brilliant  eyes  wifh 
purple  lines  and  spots  Chrysops. 

Tabanince. 

Third  joint  of  antennae  without  or  with  only  a  rudi- 
mentary basal  process  ;  thorax  and  abdomen 
with  iridescent  tomentum  ;  tibiae  dilated Lepidoselaga. 

Thorax  and  abdomen  without  iridescent  tomentum  ; 

front  of  ?  as  broad  as  long Hcematopota. 

Third  joint  of  the  antennae  with  well-developed 
basal  process. 

First  antennal  joint  short  ;  body  broad. 

Eyes  pubescent,  small  ocelligerous  tubercle  present     TJierioplecies. 

Eyes  pubescent,  but  no  ocelligerous  tubercle Atylotus. 

Eyes  bare      Tabanus. 


Fig.  71. —  Tabantis  bovinns. 


FlG.  72.  — Hanialopoia  pluvialis. 


CHRYSOPS 


177 


The  Pangonia  are  found  in  woods,  forests  and 
pastures  ;  their  flight  is  rapid.  The  proboscis  may  be 
greatly  elongated,  even  to  three  times  as  long  as  the 
body,  so  that  they  can  pierce  through  even  thick  clothes. 
An  epizootic  of  anthrax  in  Pine  Islands,  New  Caledonia, 
was  traced  to  this  genus.  The  genus  Tabanus  (fig.  49) 
is  world-wide  ;  the  short,  thick,  salient  proboscis  and 
the  last  joint  of  the  antennae  being  annulated  and  notched 
in  crescentic  form,  and  their  large  size  render  them  easily 


Fig.  73. — Head  of  Hcetnatopota. 


Fig.  74. — Wing  of  HcBmatopota  pluvialis. 


identifiable.  The  genus  Hcematopota  (fig.  72)  has  no 
crescentic  antennal  notch  (fig.  73),  and  the  wings  (fig.  74) 
overlap  ;  the  abdomen  is  also  narrower  than  in  Tabanus, 
and  the  wings  have  hyaline  spots.  The  second  joint  of 
the  antennae  is  very  short. 

The  genus  Chrysops  can  usually  be  told  by  their  wings 
(fig-  75)  being  marked  with  dark  areas  and  their  eyes 
with    purple   lines   and    spots.      They  bite   severely  and 


12 


178 


BRACHYCEKA    VERA 


usually  attack  round  the  eyes.  An  example  of  Lepido- 
sclaga  is  the  Motuca  fly  of  Brazil,  which  causes  deep 
wounds. 

Brachycera  vera. 

Family  Asilid^:  (Robber-flies). — Mostly  large  flies, 
usually  more  or  less  elongated  in  form,  and  often 
thickly  hairy  and  with  strong  bristles.  Head  broad 
and  short  with  a  freely  movable  neck ;  eyes  separate 
in    both   sexes,   with    a  deep  notch  between.     Antennae 


FlG.  75. — Chrysops  distinclipeiuiis. 

composed  of  three  segments,  the  third  elongated,  generally 
simple,  with  or  without  a  terminal  style  or  bristle  (fig. 
56-3)  ;  style  sometimes  thickened  and  forming  one  or 
two  apparent  antennal  joints.  Proboscis  firm  ;  upper  lip 
horny,  used  for  piercing;  labella  not  fleshy.  Legs  strong 
and  bristly.     Wings  when  closed  lying  parallel  over  the 


ASILIDvE — LEPTID^E 


179 


abdomen  ;  three  long  basal  cells,  two  or  three  sub- 
marginal  cells  and  five  posterior  cells  ;  third  longitudinal 
vein  forked.  These  flies  usually  feed  upon  insects. 
Some  attain  as  much  as  two  inches  in  length.  The  larvae 
live  in  rotten  wood  and  in  the  soil,  and  feed  upon  other 


FlG.  76. — Leptis  scolopacea. 


larvae.     There  do  not  seem  to  be  any  authentic  records 
of  these  Robber-flies  biting  man,  but  some  of  the  larger 
tropical  species  are  said  to  do  so;  animals  are  also  attacked 
by  them.     There  are  over  150  genera  in  this  family. 
Family  LEPTlDiE.— This  family  includes  a  number  of 


180  EMPIDIDjE 

elongated  flies  of  moderate  or  large  size  (fig.  76).  The 
veins  of  the  wings  distinct,  not  crowded  anteriorly; 
third  longitudinal  vein  forked,  basal  cells  large  ;  five 
posterior  cells  usually  present.  Third  joint  of  antennae 
complex  or  simple,  with  or  without  a  terminal  or 
dorsal  arista  or  a  terminal  style.  One  genus  only 
(SyuipJioroiiiyia)  bites,  the  rest  being  predaceous  upon 
insects.  The  section  Leptina,  in  which  the  biting  genus 
occurs,  has  short  antennae  with  simple  third  joint,  with 
a  terminal  or  dorsal  arista  or  a  terminal  style ;  the  pro- 
boscis is  short,  and  some  or  all  the  tibiae  have  spines. 
The  larvae  live  in  the  earth  and  in  decaying  wood,  sand, 
water,  and  the  nests  of  wood-boring  beetles ;  they  are 
predaceous ;  usually  cylindrical,  and  may  have  fleshy 
abdominal  legs  ;  the  anal  segment  has  a  transverse  cleft, 
and  often  two  posteriorly  directed  processes,  and  two 
stigmata  between  them.  The  genus  Symphoromyia  has 
a  single  spur  on  the  third  tibiae  ;  the  third  joint  of 
antennae  kidney-shaped,  and  the  arista  nearly  dorsal. 


FlG.  77. — Wing  of  Empis. 

Family  Empidid^e. — This  family  is  a  large  one  and 
includes  many  genera.  The  flies  have  a  piercing 
mouth,  being  all  predaceous,  feeding  upon  other  insects. 
Probably  some  attack  man  in  the  Tropics.  They  are 
mostly  small  to  moderate-sized  species,  with  small  head, 
provided  with  either  a  short  or  long  proboscis.  The 
proboscis  (fig.  57)  consists  of  two  stylets  (c),  a  hypo- 
pharynx  (d),  and  an  upper  (/)  and  lower  lip  (a).  The 
antennae  three  jointed,  the  first  two  joints  often  small, 
third  joint  very  variable,  with  or  without  a  terminal  arista 
or  style.     Abdomen  of  from  five  to  seven  segments  ;  male 


CYCLORRHAPHA 


181 


genitalia  very  prominent.  The  legs  are  of  peculiar  struc- 
ture, the  femora  thickened  and  spiny ;  metatarsi  flattened. 
Neuration  (fig.  77)  of  wing  variable  ;  there  are  three  or 
four  posterior  cells ;  the  anal  cell  is  closed  remote  from 
the  border,  sometimes  wanting,  at  other  times  it  is  closed 
near  the  border  (Hilarimorpha)  ;  then  the  discal  cell  is 
wanting.  Tegulae  small.  The  larvae  are  cylindrical,  with 
small  ventral  swellings  on  the  mesothoracic  segments  ; 
they  live  in  earth  and  amongst  decaying  vegetable  matter. 
The  pupae  have  two  points  at  the  anterior  end. 

CYCLORRHAPHA — ASCHIZA. 

This  section  does  not  contain  any  members  that  bite 
man,  but  some  are  the  agents  of  intestinal  myiasis,  e.g., 
rat-tailed  larvae  of  syrphidae,  and  phoridas  (fig.  77A). 


Wing  of  Syrphid. 


Fig.  77A. 


Wing  of  Phorida. 


The  chief  family,  the  Syrphidce,  or  Hover-flies,  are 
noted  for  the  good  some  of  their  larvae  do  in  destroying 
Aphides. 

CYCLORRHAPHA — SCHIZOPHORA. 

I.  MUSCID.E  Acalyptrat^e. — Mostly  small  flies  with 
the  antennae  composed  of  three  segments  bearing  a  non- 
terminal bristle ;  halteres  never  covered  by  a  squama 
or  basal  scale  ;  nervuration  of  wings  simple,  few  cells. 

This  group  contains  a  large  number  of  sub-families. 
None  annoy  man  to  any  noticeable  extent.  The  follow- 
ing families  are  of  economic  importance,  agriculturally 
and  otherwise  :  Chloropidce,  Trypetidce,Psilidce  (as  vegetable 
feeders),  Scatophagidcv  (dung- flies). 


Fig.  78. — Dermatobia  noxialis. 


Fig.  79  —Dermatobia  noxialis.     a  and  B,  Larvae  ("bots");   c,  puparium. 

(After  Brauer.) 


MUSCID.E    CALYPTK.K  183 

II.  Muscid^:  Calyptfl-e. — Halteres  concealed  by  a 
squama,  or  large  transparent  scale  (fig.  59). 

Family  OESTRID/E. — (Warble-flies)  (fig.  78). — Flies  of 
large  size,  thick-set,  and  often  very  hairy.  Mouth  small 
parts  rudimentary,  palpi  usually  wanting ;  eyes  rather 
small,  bare.  Head  large  ;  the  antennae  small,  composed 
of  three  segments,  more  or  less  hidden  ;  arista  simple  or 
plumose.  Thorax  broad,  with  distinct  transverse  suture. 
Abdomen  short  and  thick.  Legs  of  moderate  length,  the 
hind  pair  often  longer  than  the  rest.  Wings  with  or 
without  markings;  anal  cell,  small  discal  cell,  may  be 
absent.  The  larvae  or  bots  (fig.  79,  A  and  B)  are  provided 
with  circles  of  spines,  two  hooked  mandibles  and  anal 
breathing  pores  ;  parasitic.  They  live  in  three  ways — 
(1)  under  the  skin,  (2)  in  nasal  and  pharyngeal  cavities, 
and  (3)  in  the  alimentary  canal.  Man  as  well  as  animals 
may  be  attacked  {Dermatobia).  The  larvae  of  Dermatobia 
(fig.  79)  live  under  the  skin  of  man,  apes,  cattle,  dogs,  &c. 
In  the  adult  Dermatobia  the  arista  is  plumose  on  the 
upper  side  and  the  tarsi  slender  ;  the  proboscis  is  bent  at 
the  base  and  is  concealed  in  the  buccal  cavity  ;  squamae 
large  ;  first  posterior  cell  closed ;  body  hairy.  Larvae 
club-shaped,  slender  posteriorly,  and  surrounded  with 
rows  of  prickles  on  the  borders  of  the  segments  of  the 
apical  half.  The  chrysalis  stage  is  formed  in  a  hard 
puparium  case  (C).  The  common  species,  D.  noxialis 
(Goudot),  occurs  from  Mexico  to  Brazil,  and  is  known  as 
the  "macaw  worm,"  "  ura,"  "  torcel,"  and  "moyoquil 
worm." 

Family  SARCOPHAGID.E  (Flesh-flies).  Usually  thick 
set  and  of  variable  size.  Abdomen  composed  of  four 
visible  segments,  with  bristles  which  are  confined  to  the 
anal  end,  but  sometimes  elsewhere.  Arista  plumose  to 
the  middle,  apex  always  bare.  Some  are  metallic  (Cyno. 
uiyia).  Larvae  feed  on  decaying  animal  and  vegetable 
matter,  and  may  live  as  parasites  in  the  flesh  of  animals 
and  in  the  orifices  of  man,  also  in  wounds  and  ulcers. 
Those  of  Sarcophaga  often  occur  in  wounds  in  man,  and 


1 84  MUSCID.-E 

are  sometimes  produced  alive.  The  larvae  are  rounded, 
and  thin  anteriorly  ;  abdominal  segments  distinct,  each 
with  a  circle  of  spines  ;  mouth  with  two-curved  mandi- 
bles ;  posterior  stigmata  placed  in  a  deep  cavity,  and 
there  are  two  pointed  anal  swellings.  The  pupa  lies  in 
a  brown  oval  puparium. 

The  genus  Sarcophaga  (Meigen)  has  the  first  posterior 
cell  open;  the  tibiae  with  a  few  bristles;  the  mid  and 
posterior  cross-vein's  nearly  in  the  same  line. 

Sarcophaga  caruaria,  the  common  British  flesh-fly,  may 
be  taken  as  an  example. 

Cynomyia  (Desvoidy)  has  a  metallic  coloured  abdomen 
and  the  tibiae  with  short  hairs. 

Cynomyia  mortuorum  is  a  bright  blue  fly  aboui  the  size 
of  a  blow-fly,  and,  like  it,  lays  its  eggs  in  decaying  animal 
matter,  and  may  possibly  do  so  on  wounds. 

Sarcophila  (Rondani),  like  others  in  the  Sarcopliagidcc, 
are  viviparous.  The  females  deposit  their  larvae  in 
wounds  in  animals  and  man. 

The  larvae  of  the  genus  Ochromyia  are  also  parasitic 
under  the  skin  of  animals  and  man — Cayer  or  Senegal 
fly  (0.  anthropophaga). 

Family  Muscid^e  (House-flies,  Tsetse-flies,  &c). — A 
large  family,  easily  told  from  the  former  by  the  arista 
being  plumose  at  the  tip  (now  and  then  it  is  bare)  ;  there 
are  no  bristles  on  the  abdomen  except  at  the  tip,  and 
the  first  posterior  cell  is  very  narrow.  The  eyes  of  the 
3  contiguous,  bare  or  hairy  in  both  sexes.  Abdomen 
composed  of  four  visible  segments.  This  family  con- 
tains the  house-fly  (Mnsca),  blue-  and  green-bottle  flies 
(Liicilia  and  Calliphora),  stable  or  "stinging  flies" 
(Stomoxys),  horse-flies  (H  can  1  at  obi  a),  and  tsetse-flies  (Glos- 
siua.  The  larvae  are  variable  ;  some  live  in  decaying  vegeta- 
tion, in  decaying  animal  matter  and  faeces  ;  others,  as  the 
screw-worm  (Chrysomyia),  as  parasites  in  animals  and 
man  :  so  also  may  Calliphora  and  Luc  Hi  a.  The  Stomoxy- 
iucu,  which  include  the  stable-fly,  tsetse-fly  and  the  horn- 
fly,  have  elongated,  piercing  probosces,  and  are  blood- 
suckers. 


STOMOXYS  185 

The  following   characters  will    separate  the  more  im- 
portant genera  : — 

Proboscis  long,  used  for  piercing  ;  palpi  shorter  than 

proboscis  Stomoxys. 

Palpi  nearly  as  long  as  proboscis     Hcematobia. 

Proboscis  very  long,  straight    Glossina. 

Proboscis  short,  not  adapted  for  piercing  ;  arista 
plumose  on  both  sides  ;  curvature  of  fourth  vein 
angular ;  mid  tibiae  without  bristles  on  inner  side  ; 
abdomen  non-metallic  ;  blackish  species  with 
more  or  less  yellowish  markings  Musca. 

Mid  tibiae  with  bristles  on  the  inner  side  ;  abdomen 
&c,  with  metallic  colom-s. 

Thorax  blackish  Calliphora. 

Thorax  black  with  whitish  longitudinal  stripes,  more 

or  less  metallic Chrysomyia. 

Thorax  unicolorous,  metallic    Lucilia. 


Fig.  81. — Wing  of  Stomoxys  calcitrans. 

Genus  Stomoxys. 

In  the  genus  Stomoxys  the  solid,  elongate  proboscis, 
jointed  at  an  angle  near  its  base,  is  the  obvious  char- 
acteristic. The  type  species  of  the  genus  is  Stomoxys 
calcitrans,  which  is  very  like  the  common  house-fly  in 
general  appearance. 

Month-parts  in  Stomoxys. — The  proboscis  beyond  the 
angle  projects  horizontally.  The  palpi  are  very  slender 
and  scarcely  one-third  the  length  of  the  proboscis  ;  they 
spring  from  above  the  angle  and  are  not  in  contact  with 
the  proboscis. 

The  proboscis  consists  of  three  parts — labium,  labrum 
and  hypopharynx. 


1 86 


STOMOXYS 


The  labium  is  the  only  part  seen  in  the  ordinary  resting 
position.  It  is  a  solid-looking  chitinous  structure,  ovoid 
in  cross-section,  with  a  narrow,  shallow  groove  on  its 
anterior  or  upper  surface,  within  which  lie  the  labrum 
and  hypopharynx.  The  lateral  walls  of  the  labrum  are 
incurved  below  to  form  a  tube,  with  a  slit  along  the  lower 
side.  This  slit  is  closed  by  the  apposition  of  the  hypo- 
pharynx,  a  delicate  rod  within  which  lies  the  excretory 
tube  of  the  salivary  ducts. 


FlG.  82. — Cross-section   of  proboscis   of   Stn/noxys  (after  Giles).       h,   Hypo- 
pharynx  ;  /,  labium  ;  Inn,  labrum  ;  m,  muscle  ;  /,  trachece. 

Dissection. — To  expose  the  structures  in  the  thorax  in 
such  flies,  snip  off  with  sharp  scissors  the  legs  with  a  little 
of  the  ventral  wall  of  the  thorax.  There  will  then  be  seen 
a  number  of  vertical  bundles  of  muscle  fibre  (the  coxal 
muscles).  Separate  these  muscle  bundles  in  the  middle 
line  and  expose  the  great  thoracic  ganglion,  a  sausage- 
shaped  mass  of  considerable  size  situated  opposite  the 
first  pair  of  legs.  Clean  this  away,  and  just  under  its 
anterior  end  will  be  seen  a  nodule  about  the  size  of  a 
small  pin's  head;  this  nodule  is  the  proventriculus.  Ex- 
tending backwards  from  it  is  a  glistening  tube,  the  mid- 


DISSECTION    OF   STOMOXYS  187 

gut,  and  lying  upon  it  three  delicate  tubes,  the  crop  duct 
centrally  placed,  and  at  the  sides  the  commencement  of 
the  salivary  glands. 

The  abdominal  viscera  can  be  exposed  by  snipping 
through  the  chitinous  covering  of  the  dorsum  in  a  longi- 
tudinal direction  and  floating  the  contained  structures 
in  normal  saline  solution. 


Fig.  83. — Dissections  of  the  abdomen  of  Stomoxys,  after  Lieut.  Tulloch, 
seen  from  above,  with  the  alimentary  canal  unravelled.  CD,  Common 
seminal  duct  ;  D,  seminal  duct  ;  MT,  Malpighian  tubes  ;  O,  junction  of  distal 
intestine  and  mesenteron;  R,  rectum;  rp,  rectal  papillae;  SG,  salivary 
glands  ;  T,  testis ;  TT,  dilated  ends  of  left  Malpighian  tubes ;  vs,  vesiculad 
seminalis. 

Internal  Anatomy. — The  buccal  cavity,  which  is  a 
narrow  tube,  is  contained  in  the  base  of  the  proboscis. 
From  it  the  pharynx  runs  almost  vertically  upwards  in 
the  head,  then  bends  sharply  backwards  to  become  the 
oesophagus,  the  latter  being  continued  backwards  as  the 
mid-gut.  At  the  junction  of  the  oesophagus  and  mid- 
gut is  situated  the  proventricul.us,  into  which  opens  also 
the  duct  of  the  crop,  a  large  hollow  sac  situated  in  the 


lob  DISSECTION    OF   STOMOXYS 

anterior  part  of  the  abdomen  between  the  mid-gut  and 
the  salivary  glands.  The  mid-gut  runs  backwards  into 
the  abdomen  as  a  narrow  tube  until  it  reaches  nearly  to 
the  posterior  border  of  the  crop,  where  it  becomes  dilated. 
This  dilated  portion  has  three  simple  coils,  which  lie 
superposed  in  the  middle  of  the  abdomen.  The  tube 
then  gradually  narrows  and  into  this  region  the  four 
Malpighian  tubes  open.  The  alimentary  canal  is  con- 
tinued as  a  uniformly  narrow  tube  to  the  rectum.  The 
narrow  lower  intestine  has  variable  bends  but  is  not 
coiled.  The  rectum  is  a  dilated  cone-shaped  cavity  with 
its  apex  towards  the  anus;  within  it  are  four  rectal  papillae. 
Below  the  dilatation  the  rectum  is  continued  as  a  shoit 
narrow  tube  to  the  anus.  The  appendages  of  the  alimen- 
tary canal  are  the  Malpighian  tubes,  the  crop  and  the 
salivary  glands. 

The  salivary  glands  lie  in  the  abdomen  ventral  to  the 
crop,  they  are  continued  forward  into  the  thorax  and 
become  the  salivary  ducts  at  the  anterior  end  of  the 
thorax.  These  ducts  join  in  the  head,  forming  the 
common  salivary  duct  which  passes  into  the  hypopharynx. 

In  the  genus   Hcemaiobia   the  proboscis  is  similar  to 
that  in  Stomoxys,  but  the  palpi  at  once  separate  it.    Hcema- 
iobia serrata,  the  "horn-fly"  of  North  America,  causes 
much  annoyance  to   cattle  and  bites  man,  but  in  Great. 
Britain  it  seems  harmless. 

Genus  Glossina. 

In  the  genus  Glossina  the  proboscis  is  long  and  straight. 
Palpi  are  the  same  length  as  the  proboscis  and  form  a 
sheath  for  it.  Arista  plumose  to  the  tip,  the  hairs  being 
on  upper  surface  only  and  compound.  The  wings  when 
at  rest  are  crossed  over  one  another  "  scissors  like,"  and 
project  well  beyond  the  abdomen.  The  wing  venation 
is  characteristic,  especially  in  the  course  of  the  fourth 
longitudinal  vein,  which  makes  two  bends,  one  before  it 
meets  the  anterior  transverse  vein  and  another  before  it 
reaches  the  costal  margin. 


GLOSSINA 


189 


The  females  of  this  genus  produce  their  larvae  full 
grown,  the  larvae  changing  to  pupae  without  feeding. 
The  larvae  are  deposited  in  the  neighbourhood  of  decay- 
ing vegetation,  particularly  about  the  roots  of  certain 
trees,  such  as  the  banana  tree  and  others  of  its  class,  and 
burrow  into  this  before  pupating. 


Fig.  84. — Transverse  section  of  the  proboscis  of  Glossina  palpalis  at  almost 

mid-length. 


Fig.  85.  —  Glossina  morsitans. 

Glossina  are  found  in  belts  usually  in  the  vicinity  of 
streams  or  rivers  on  the  edge  of  forest  land.  In  such 
situations  they  may  be  present  in  large  numbers  for  a 
few  hours,  at  other  times  none  or  very  few  can  be  found. 


190  GLOSS1XA 

Mouih-parts  in  Glossina. — The  maxillary  palpi  project 
horizontally  and  being  grooved  on  the  inner  aspects  act 
as  ensheathing  organs  for  the  proboscis. 

The  proboscis  is  expanded  at  the  base,  the  remainder 
being  proportionately  very  slender;  it  reaches  almost  to 
the  end  of  the  palpi  and  is  slightly  curved,  with  the  con- 
vexity beneath.  It  consists  of  three  parts — labium  or 
lower  lip,  labrum  or  upper  lip,  and  the  hypopharynx. 

The  labium  consists  of  a  large  basal  bulb  and  a 
terminal  long,  slender,  chitinous  rod.  It  is  deeply 
grooved  on  its  upper  surface  and  bent  upwards  and 
inwards. 

The  labrum  is  a  narrow  chitinous  rod,  the  prolonga- 
tion of  the  exterior  wall  of  the  pharynx.  Its  edges  are 
turned  downwards  and  inwards,  so  that  it  forms  about 
two-thirds  of  a  tube.     It  terminates  in  a  sharp  point. 

The  Jivpopharvnx  is  a  solid  rod,  semilunar  in  cross- 
section,  with  a  rib  running  down  its  ventral  surface, 
within  which  is  the  salivary  canal. 

As  will  be  seen  from  the  figure,  the  labium  and  labrum 
together  constitute  a  tube  up  which  the  blood  passes  to 
the  pharynx.  The  convex  dorsal  surface  of  the  hypo- 
pharynx  fits  closely  to  the  sides  of  the  labrum. 

The  internal  anatomy  in  Glossina  is  in  general  very 
similar  to  that  in  Stomoxvs.  The  mid-gut  is,  however, 
longer  and  larger. 

Synoptic  Tabic  of  Species. 

I.  Hind  tarsi  dark,  or  at  least  all  the  segments  more  or 
less  dark  (in  the  ?  of  G.  tachinoides  the  basal  half 
of  the  first  joint  and  the  extreme  bases  of  the  other 
segments  are  usually  pale). 

(1)  Ground  colour  of  abdomen  ochraceous  buff, 
with  interrupted  dark  brown  deep  transverse 
bands  and  sharply-defined  pale  hind  borders 
to  the  segments,  a  very  conspicuous  square 
or  oblong  pale  area  in  the  centre  of  the 
second  segment ;  small  species,  not  exceed- 
ing 8  mm.  in  length,  exclusive  of  proboscis, 
the  males  much  smaller   tachinoides. 


GLOSSINA  191 

(2)  Abdomen  very  dark,  or  for  the  most  part 
uniformly  brown,  hind  borders  of  segments 
if  lighter  extremely  narrow  and  cinereous ; 
pale  area  in  centre  of  second  segment 
usually  triangular,  with  the  apex  directed 
backwards  and  continued  into  a  cinereous 
median  stripe  ;  larger  species. 

{a)  Third  joint  of  antennas  dusky  brown 
to  cinereous  black. 

(a)  Thorax,  pleurae  and  coxae  more 
or   less    uniform  in  colour   or 

with  stripes  only ftalpalis. 

(/3)  Thorax  with  elongated  trans- 
verseblackspots  ;  pleurae, coxae 
and  femora  with  conspicuous 

black  spots  metadata. 

(b)  Third  joint  of  antennae  pale  (orange- 
buff)  ftaUicera. 

II.  Hind  tarsi  not  entirely  dark  ;  last  two  joints  alone 
dark,  remainder  pale. 

A.  Smaller  species;  length  rarely  reaching  11 
mm.,  often  considerably  less  ;  wing  expanse 
not  exceeding  25  mm. 

(a)  Last  two  joints  of  front  and  middle 
tarsi  with  sharply  defined  dark  brown 
or  black  tips. 
Generally    distinctly    larger ;    head 
wider ;    front   darker   and   nar- 
rower    in     both     sexes,     sides 
parallel  in  $  ;  abdominal  bands 
deeper,   leaving   hind    margins 
only   narrowly    pale ;      hypopy- 
gium  in   $■  smaller,  darker  and 
more   hairy ;    tip    of    abdomen 
more   thickly   clothed    laterally 
with  short  black  hair,  bristles 
on  sixth  segment  finer  and  less 

prominent   longipalftis. 

Usually  smaller ;  head  narrower  ; 
front  paler  and  wider;  eyes  in 
S  as  well  as  in  5  distinctly 
converging  towards  vertex  ; 
abdominal  bands  less  deep  ; 
pale  hind  margins  of  segments 


[92 


LUCILIA 


therefore  deeper  ;  hypopygium 
in  the  J  larger,  paler,  some- 
what more  oval  in  outline,  and 
clothed  with  fewer  fine  hairs  ; 
tip  of  $  abdomen  less  hairy 
laterally ;  bristles  on  the  sixth 
segments    in     3      stouter    and 

more  conspicuous morsitans. 

(l>)  Last  two  joints  of  front  and  middle 
tarsi  without  sharply-defined  black  or 
brown  tips :  front  and  middle  tarsi 
entirely  yellow,  or  last  two  joints  of 
former  faintly  tipped  with  pale  brown  fiallidipes. 
B.  Large  species  ;  length  at  least  1 1  mm.,  wing 
expanse  at  least  25  mm. 

Dorsum  of  thorax  with  four  sharply-de- 
fined small  dark  brown  or  oval  spots 
arranged  in  a  parallelogram,  two  in 
front  of  and  two  behind  transverse 
suture  ;    bulb  at  base  of  proboscis 

brown  at  the  tip longipennis. 

Dorsum  of  thorax  without  such  spots, 
though  with  more  or  less  distinct 
longitudinal  stripes  ;  bulb  at  base  of 
proboscis  not  brown  at  the  tip fusca. 


Fig.  86. — Litcilia  tasar. 


Genus  Lucilia  (fig.  86),  the  so-called  green-bottle  flies, 
have  a  soft  proboscis  (fig.  87).     They  are  all  of  metallic 


AUCHMEROMYJA 


193 


colour  and  the  abdomen  is  short  and  round  ;  the  third 
segments  of  the  antennas  are  quadruple  the  size  of  the 
second.     Basal    half   of    third    longitudinal    vein    carries 


Fig.  87. — Head  of  Lucilia  azsar. 


/ 


Fig.  S8. —  Chrysomyia  macellaria. 

spines.     The  ova  and  larva?  are  often  deposited  on  wounds 
and  ulcers  in  animals  and  man  (L.  sericata).     This  species 
causes  the  well-known  "maggot"  in  sheep. 
13 


194  AUCHMEROMYIA 

Genus  Chrysomyia  (Compsornyia). — This  genus  also 
contains  metallic-coloured  Hies  which  differ  from  Lucilia 
in  that  the  thorax  is  striped.  The  screw-worm  fly  (C. 
macellaria,  fig.  88)  is  found  in  North  and  South  Ameri<  ;i 
and  the  West  Indies,  hut  does  not  attack  man  farther 
north  than  Kansas. 


Fir. .  89. — Anchmeromyia  luleola. 

Genus  Auchmeromyia. —  The  blood-sucking  larva  of 
one  species,  A.  luteola,  has  been  described  under  the 
name  of  "The  Congo  Floor  Maggot."  The  perfect  in- 
sect is  about  11  mm.  in  length,  of  a  pale  yellow  colour. 
The  head  is  broad,  equal  in  width  to  the  thorax,  and  the 
proboscis  lies  in  a  deep  groove.  Eyes  in  both  $  and  ? 
widely  separate.  The  dorsum  of  the  thorax  is  flattened 
and  marked  by  longitudinal  dark  stripes.  The  abdomen 
consists  of  five  segments,  the  second  being  much  the 
longest  and  broadest.  This  segment  is  dark  brown  or 
black  in  its  posterior  half,  the  anterior  half  being  pale 
and    bounded   in    front  bv   a   dark    narrow    line    on    the 


ANTHOMYID.E  195 

posterior  edge  of  the  first  segment.  The  third  and  fourth 
segments  are  dark  brown  to  black  in  colour.  The  fifth 
segment  is  small  and  contains  the  genitalia. 

The  larva  is  dirty  white  in  colour,  about  15  mm.  in 
length,  the  body  comprising  eleven  segments.  The 
anterior  segment  is  conical  in  shape  and  carries  the 
mouth,  which  is  armed  with  two  black  hooklets  and 
paired  teeth.  They  live  under  mats  or  in  the  cracks  of 
the  earthern  floors  of  native  huts.     They  feed  at  night. 

This  fly  has  a  wide  distribution  from  Northern  Algeria 
to  Natal,  and  is  especially  common  in  the  Congo  and 
about  Lake  Chad. 

Genus  Cordylobia. — The  larva  of  one  species  (C.  anthro- 
pophaga)  lives  beneath  the  skin  of  man  and  animals, 
and  feeds  on  the  tissues.  The  fly  itself  closely  resembles 
Auchmeromyia  luteola,  but  differs  from  it  in  that  the 
second  segment  of  the  abdomen  is  not  so  large  or 
clearly  defined,  and  the  eyes  are  much  closer  together, 
those  in  the  male  nearly  meeting  in  the  middle  line. 
Widely  distributed  in  Africa. 

Family  Anthomyid^e. — These  are  mostly  moderate- 
sized,  dull-coloured  flies  resembling  the  common  house 
fly.  The  arista  is  plumose,  pubescent,  or  bare.  Abdo- 
men composed  of  four  or  five  segments  ;  sometimes  there 
are  no  bristles  on  the  body,  but  they  are  usually  present. 
The  first  posterior  cell  of  the  wings  broadly  open  ;  tegul?e 
of  considerable  size.  Male  eyes  usually  contiguous. 
It  is  closely  connected  on  one  hand  with  the  Muscidce, 
and  on  the  other  with  the  Sarcophagidce.  None  are 
metallic.  The  open  first  posterior  cell  is  the  chief 
character.  The  following  genera  have  been  connected 
with  man  either  as  parasites  or  by  causing  other  annoy- 
ance— viz.,  Hydrotcva  (Desvoidy),  Homalomyia  (Bouche), 
and  Hylemyia  (Desvoidy). 

They  may  be  told  as  follows  : — 

Eyes  of  $  close  together  ;  tegula  large,  larger 
than  ante-tegula  ;  fore  femora  of  $  with 
processes,  tubercles,  &c,  below  ;  arista 
always  somewhat  pubescent  ;  eyes  bare ; 
black  or  blue-black,  and  pilose  ...         ...    Hydrotcea  (Desvoidy). 


196 


HOMALOMYIA 


Eyes  of  <?  close  together,  bare  ;  tegula  large  ; 
abdomen  nearly  bare,  unspotted  :  head 
almost  composed  of  eyes  ;  antenna;  short 
third  joint  elongated;  arista  bare;  mid- 
legs  of  $  often  with  peculiar  structures  ; 
black  and  grey  Homalomyia  (Bouchei. 

Arista  plumose  ;  eyes  bare  ;  elongated  species, 

grey  or  black Hylemyia  (Desvoidy;. 

The  genus  Homalomyia  (fig.  90)  has  often  occurred 
in  human  beings  in  the  larval  state  in  the  intestines, 
being  passed  alive  in  the  faeces.  Most  larvae  in  this 
family  are  vegetable  feeders.     They  are  normally  slender 


Fig.  90. — Homalomyia  canicularis. 


and  cylindrical,  or  flat  and  oval,  with  four  rows  of 
thread-like  processes  on  the  segments,  and  have  two 
mouth  hooks.  The  puparium  may  be  oval  or  flattened. 
In  Homalomyia  they  have  curious  branched  processes 
(fig.  91).     The  genus  Hydrotaia  also  occurs  in  the  larval 


TACHINID/K.    PUPIPARA 


197 


form  in  human  beings.  The  characteristic  neuration  is 
shown  in  iig.  92.  Hylemyia  larvae  have  also  occurred  in 
human  excreta,  having  been  passed  per  auum.  Some 
are  dung  frequenters  and  produce  living  young. 


Fig.  91. — Larva  of  Homalomyia, 


Fig.  92. — Wing  of  Hydrotcea  ciliata. 

Family  Tachinid^e. — Like  Anthomyidce,  but  always 
bristly.  Arista  bare.  Palpi  formed  of  one  segment.  All 
veins  of  the  wings  simple  ;  basal  cells  large  ;  three  pos- 
terior cells  ;  first  posterior  cell  closed  or  only  just 
opened.  Squamae  large.  Larvae  parasitic  in  insects, 
especially  in  the  larvae  of  certain  Lepidoptera. 


PUPIPARA. 

Blood-sucking,  parasitic  on  vertebrates  (except  Braula). 

Family     Hippoboscid.e. — Parasites    upon    birds    and 

mammals  when  mature.       Proboscis   may  be   longhand 


198 


HIPPOBOSCIIXK 


FlG.  93. — Hippobosca  equina  (enlarged  four  times). 


Fig.  94. — Melophagus  ovinus  (enlarged  twelve  times). 


NYCTERIBID/E  199 

sharp.  Palpi  absent  ;  antennae  placed  in  pits,  composed 
of  one  segment,  with  or  without  terminal  bristle  or 
hairs.  Eyes  round  or  oval,  often  very  small.  Thorax 
flat,  leathery;  scutellum  broad  and  short.  Abdomen 
leathery,  inflated,  no  sutures  visible.  Legs  short,  strong ; 
claws  large  and  dentate  ;  empodia  distinct.  Wings 
present  or  absent  (vide  figs.  93,  94).  The  larvae  are  born 
nearly  matured  in  the  puparium  case,  passing  most  of 
their  development  in  the  body  of  the  parent.  Of  general 
louse-like  form.  This  family  contains  the  forest-fly 
(Hippobosca  equina)  (fig.  93),  and  the  sheep  ked  (Melo- 
phagus  oviuiis)  (fig.  94).  The  proboscis  is  composed  of 
elongated,  hard,  closely  applied  flaps  and  an  inner  tube 
between.  They  live  on  horses,  cattle,  and  birds,  and  now 
and  then  attack  men. 

Family  Nycteribid^e. — Found  exclusively  on  bats. 
Spider-like ;  no  wings.  Eyes  and  ocelli  indistinct  or 
wanting.     Legs  long,  femora  and  tibiae  flattened. 

Two  other  families,  Braulidce  and  Streblidce,  occur;  the 
former  live  on  bees,  the  latter  on  bats. 


200 


CHAPTER    XI. 

MOSOI   ITOES. 

MOSQUITOES  are  the  hosts  of  many  parasites,  and  some 
of  these  are  injurious  to  man  or  the  lower  animals.  As 
these  diseases  include  malaria  and  yellow  fever,  at  least 
one  of  the  human  filarial,  as  well  as  the  Proteosoma  of  birds 
and  the  Filaria  immitis  so  fatal  to  dogs,  a  good  working 
knowledge  of  the  structure,  life-history,  and  modes  of 
classification  of  these  insects  is  required  for  tropical  work. 

Mosquitoes,  or  Culicidce,  belong  to  the  order  of  dipterous 
insects,  as  they  have  the  anterior  pair  of  wings  mem- 
branous, whilst  the  posterior  pair  are  represented  by  a 
pair  of  club-shaped  processes,  the  halteres  or  balancers. 

The  insect  is  divided  naturally,  into  three  regions  :  (i) 
head,  (2)  thorax,  (3)  abdomen.  To  the  head  are  attached 
the  sensory  and  biting  organs,  consisting  of  two  com- 
pound eyes,  two  antennas,  two  palpi,  and  a  complex 
suctorial  and  piercing  organ,  the  proboscis. 

To  the  thorax  are  articulated  a  pair  of  wings,  a  pair 
of  balancers,  and  three  pairs  of  legs  ;  whilst  the  abdomen 
is  segmented  and  terminates  in  the  anus  and  external 
organs  of  generation. 

In  the  Culicidce  the  head,  thorax,  abdomen  and  legs  are 
thickly  covered  with  scales  in  most  of  the  genera,  whilst 
on  the  wings  scales  are  found  only  at  the  edge  and  on 
the  veins.  The  character  and  arrangement  of  the  scales 
are  important  points  in  the  differentiation  of  genera. 
The  absence  of  scales  on  the  wings  or  the  presence  ol 
scales  on  other  parts  of  the  wings  than  those  mentioned, 
or  the  substitution   of  hairs  for  scales,  are  valuable  aids 


MOSQUITOES  201 

in  the  differentiation  of  other  insects  from  the  Culicidce. 
The  type  of  venation  of  the  wings  and  the  characters  of 
the  cephalic  appendages  are  of  value  both  for  identifica- 
tion of  the  family  Culicidcc,  and  for  differentiation  of 
genera,  species,  and  sexes. 

Scales  lose  their  colour  and  become  too  transparent 
for  proper  examination  unless  the  specimens  are  mounted 
dry,  so  that  for  identification  of  species  it  is  advisable 
that  the  mosquitoes  should  be  mounted  dry  and  so 
arranged  that  all  the  surfaces  can  be  examined.  It  is 
best  to  examine  or  send  for  examination  young  mos- 
quitoes, as  in  older  specimens  many  scales  are  rubbed 
off  and  the  insects  otherwise  injured. 

Mosquitoes  hatched  out  from  pups:  should  be  kept 
alive  for  at  least  twenty-four  hours,  as  if  killed  when  too 
young  they  become  much  distorted  when  they  dry.  They 
should  be  kept  in  a  dark  place,  as  they  then  are  less  liable 
to  have  the  scales  rubbed  off. 

The  mosquitoes  must  be  killed  rapidly,  and  a  cyanide 
pot  is  invaluable  in  this  connection,  though  chloroform 
vapour,  formalin,  or  even  tobacco  smoke,  may  be  used. 
The  dead  mosquito  must  be  mounted  without  delay,  as 
the  limbs  soon  lose  their  pliability.  To  mount  they 
should  be  placed  on  their  backs  on  a  piece  of  cork 
felt.  A  small  square  or  a  circle  of  thin  card  should  be 
taken,  and  on  one  side  of  it  the  date  and  place  of  capture 
of  the  mosquito  should  be  written,  with  a  distinguishing 
number  if  the  name  is  not  known. 

A  fine  entomological  pin  (No.  20)  should  be  taken  up 
with  forceps  near  the  point,  and  the  piece  of  card  with 
the  blank  side  downwards  should  be  placed  on  a  piece 
of  cork  felt.  The  pin,  still  held  in  the  forceps,  should  be 
pushed  through  the  card.  The  hold  on  the  pin  should 
then  be  shifted  higher,  and  the  pin  pushed  still  further 
through  the  card  till  about  half  of  it  is  through.  The 
pin,  still  held  in  the  forceps,  with  the  card  transfixed  on 
it,  should  then  be  pushed  through  the  thorax  of  the 
mosquito.     On  lifting   up  the  pin   the    mosquito,    which 


202 


MOUNTING   MOSQUITOES 


has  been  transfixed,  will  remain  on  the  pin,  and  on  turn- 
ing the  card  upside  down  the  legs  and  wings  can,  with  a 
few  touches  of  a  clean  needle,  be  arranged  so  as  to  be 
readily  visible,  and  will  not  hide  any  part  of  the  back 
of  the  insect.     A  stout  pin  should  then  be  run  through 


1 


/£ 


Fig.  95. — a,   Forceps  ;  /',   pin;  c,  disc;  d,  cork;  in,   mosquito;  e,  large  pin 

to  carry  disc. 

the  corner  of  the  piece  of  card  into  the  cork  felt  floor  of 
the  collecting  box.  To  prevent  insects  attacking  the 
specimens  some  powdered  naphthalin  enclosed  in  a  cloth 
bag  should  be  placed  in  the  box,  and  securely  fixed  to  the 
side  of  the  box  with  strong  drawing-pins,  or  melted 
naphthalin  may  be  poured  in  the  corners  of  the  box.     An 


EXAMINATION    OF   MOSQUITOES  203 

alternative  plan  is  to  pin  securely  into  the  corners  of  the 
box  some  small  pieces  of  sponge  and  on  these  to  place 
a  drop  or  two  of  creosote. 

To  examine  such  a  specimen  a  low  power,  one  inch  or 
two-thirds  of  an  inch,  is  required.  With  such  a  power 
the  character  of  the  scales  on  each  part  of  the  insect  can 
be  examined.  The  examination  should  be  made  by  re- 
flected light  and  the  insect  so  rotated  that  the  part  to  be 
examined  is  horizontal.  This  can  be  done  best  by  alter- 
ing the  inclination  of  the  large  pin  and  using  a  strip  of 
cork  felt  as  a  slide  (fig.  96).  In  this  way  each  part  of  the 
upper  surface  can  be  examined  in  succession. 


Fig.  96. 

To  examine  the  under  surface,  a  second  mosquito 
mounted  with  its  back  towards  the  card  is  required. 

The  main  types  of  scales  found  in  the  Culicidce  are 
represented  in  the  drawing  (fig.  97).* 

These  scales  can  for  descriptive  purpose  be  reduced 
to  the  small  number  of  types  represented  : — 

(a)  Broad,  flat,  spade-shaped  or  tile-shaped  scales. 

(b)  Broad,  expanded,  asymmetrical  scales. 

(c)  Narrow,  asymmetrical  scales. 

(d)  Narrow,  hair-like  scales. 

(e)  Narrow  curved  scales  or  crescents. 
(f  &  g)  Spindle-shaped  scales.    . 

(h  &  i)   Upright  fork  scales  or  darts. 
( j)  Long  twisted  scales. 
(k)   Pyriform  scales. 

*  Figs.  97  and  99—101  are  reproduced  by  kind  permission  of  the 
Editor  of  the  Journal  of  Tropical  Medicine. 


!C>4 


TYPES   OK   SCALES 


Fig.  97. — Types  of  scales,  a  to  k.  Head  and  scutellar  ornamentation,  1  to 
4  ;  forms  of  clypeus,  6.  1,  Head  and  scutellum  of  Stggomyia,  &c.  ;  2,  of 
Culex  ;  3,  of  sEdes,  &c.  ;  4,  of  Megarhinus,  &c.  ;  5,  Head  ornamentation 
of  Cellia  and  some  other  Anophelina;  6,  clypeus,  a,  of  '  Cule.x ;  /'',  of 
Stegomyia  ;   t',  of  foblotia.     (Theobald.) 


APPENDAGES  205 

On  the  wings  other  types  of  scales,  either  lanceolate, 
long,  narrow  scales  pointed  at  the  free  end,  or  long  and 
narrow  and  with  square  free  ends,  are  met  with  (fig.  101). 

Head  Appendages. — The  head  appendages  can  be  easily 
seen  in  most  specimens  mounted  as  described,  but  for 
more  minute  examination  it  is  better  to  cut  off  the  head 
and  mount  it  in  a  shallow  cell  either  as  a  dry  specimen 
or  in  glycerine  jelly.  In  this  way  the  parts  are  not  much 
distorted,  and  if  a  thin  slide  be  used  both  sides  of  the 
specimen  can  be  examined.  Canada  balsam  can  be  used, 
for  the  examination  of  the  scales,  hairs,  &c,  but  not 
satisfactorily,  as  they  become  too  transparent. 

Proboscis. — To  examine  the  component  parts  of  the 
proboscis  it  is  better  not  to  use  the  shallow  cell,  but  to 
forcibly  compress  the  head  after  soaking  in  liquor  potassae 
for  twenty-four  hours,  so  as  to  cause  the  various  com- 
ponent parts  of  the  proboscis  to  separate  ;  in  one  or  more 
specimens  all  the  elements  can  be  seen. 

Palpi. — The  points  to  be  noted  in  the  palpi  are  their 
length  relative  to  the  proboscis,  the  number  of  joints,  and 
the  colour,  shape  and  arrangement  of  scales  and  hairs. 
To  determine  the  number  of  joints  it  is  necessary  to 
remove  the  scales  from  the  palpi. 

The  Antennce. — Their  length,  and  the  relative  lengths 
of  the  different  joints.  The  number,  length  and  arrange- 
ment of  hairs,  and  the  presence  or  absence  of  scales. 

The  different  regions  of  the  mosquito  are  shown  in  the 
diagram  (fig.  98).  To  the  head  are  attached  the  append- 
ages already  mentioned,  and,  in  addition  to  these,  the 
back  part  of  the  head,  or  occiput,  requires  close 
examination. 

The  thorax  is  composed  of  three  segments  fused 
together.  The  greater  part  is  formed  by  the  second 
segment,  or  mesothorax.  Anteriorly  on  each  side  are 
two  rounded  projections,  the  prothoracic  lobes,  the  rem- 
nants of  the  anterior  segment.  The  posterior  edge  of 
the  mesothorax  is  a  narrow,  overhanging,  often  trilobed 
plate — the  scutellum.  The  scales  on  this  part  of  the  thorax 
are  of  generic  value. 


206 


ANATOMY   OF   MOSQUITO 


Probosc 


Antennae 


!  Wing  scales""! ^ 


Palpi 

Eyes'' 
Occiput 

Pro  thoracic' 

lobes 
Mesothorax---" 

Scutellum 
Meta  thorax"" 

orMetanotunO// 

First  abdominal 
segment 

Abdomen  — 


C 


Basal 
lobes  of  Q 


Basa 
lobes.. 


asper. 


Proboscis 

Palpi 

Antennae 

Basal  lobes  ofanlennae 

Frons 

Vertex 

Eyes 

Occiput 
Nape 


4[?  tarsal 


Llni.clS.5.3 


5^  tarsal 


Fig.  98.     (After  Tlieubald.) 


METANOTUM 


207 


Partly  overlapped  by  the  scutellum  is  a  rounded  mass 
connecting  the  thorax  and  abdomen,  known  as  the  meta- 
thorax  or  metanotum.     This  is  the  third  segment  of  the 


Fig.  99. — Types  of  metathorax  (Theobald),     a,  Culicina;  b,  Dendro- 
t/iyina;  c,Joblotina. 

thorax.     On  each  side  of  the  metathorax  are  the  halteres, 
which  arise  from  the  sides  of  the  mesothorax. 

The  abdomen  is  segmented  and  has  no  lateral  append- 


208  THORAX   AND   ABDOMKX 

ages.  The  last  segment  terminates  in  the  external 
genitalia.  These  are  of  specific  but  rarely  of  generic 
value, 

Thorax  and  Abdomen. — In  the  examination  of  the  dry 
mounted  specimen  by  this  method,  each  part  of  the 
mosquito  should  be  examined  in  turn.  By  altering  the 
angle  in  the  manner  described,  the  different  parts  repre- 
sented in  the  diagram  can  all  be  clearly  made  out  and 
the  character  of  the  scales  covering  these  parts  investigated. 

Metathorax. — It  is  well  to  first  examine  the  metathorax 
or  metanotum.  This  part  is  nude  in  all  sub-families 
except  Dendromyince,  in  which  there  are  hairs,  in  Joblotincc 
and  Limatus,  which  have  both  hairs  and  scales  (fig.  99). 

Scutellum. — Overhanging  the  metathorax  is  the  scutel- 
lum,  bordered  by  a  row  of  stiff  hairs  and  covered  with 
scales.  These  scales  are  not  necessarily  of  the  same  type 
as  those  covering  the  thorax,  but  are  often  the  same  as 
the  scales  covering  the  middle  of  the  occiput,  with  the 
exception  that  there  are  no  upright  fork-scales.  The 
scales  on  the  scutellum  are  of  great  generic  importance 
(fig.  97),  and  the  shape  of  the  edge  is  of  similar  value  in 
separating  the  AnopJieliiuc  and  Megarhinina  from  the 
other  Culicidcc. 

The  character  of  the  scales  on  the  abdomen  and 
thorax  are  used  by  Mr.  Theobald  to  subdivide  the  old 
genus  of  Anopheles,  and  in  the  further  subdivision  of  the 
CulicincE. 

Occiput. — The  scales  on  the  occiput  should  next  be 
examined.  They  vary  according  to  genera.  The  upright 
fork  scales  (//  and  i)  are  found  only  in  this  situation.  In 
some  genera  no  other  scales  are  found  on  the  head.  In 
most  genera  the  scales  at  the  side  of  the  occiput  are  tile- 
shaped  scales  (a),  but  whilst  in  some  genera  (Megarhinina) 
these  scales  extend  all  over  the  middle  line  of  the  occiput, 
and  are  the  only  scales  found,  in  others,  as  Stegomyia, 
they  are  found  also  with  fork-scales;  in  others,  again, 
they  are  not  found  in  the  middle  of  the  occiput,  but  are 
replaced  by  spindle  scales  (/),  either  alone  as  in  /Edes,  or 


WINGS 


209 


with  narrow-curved  and  upright  fork-scales  as  in  Culex, 
Mansonia,  &c.  (tig.  97)., 

Wings. — The  type  of  wing  venation  can  be  seen  in  a 
specimen  mounted  as  described  above,  but  is  better 
seen  in  the  wings  when  detached,  flattened  out  and 
examined  dry.  The  character  of  the  scales  covering  the 
longitudinal  veins  on  the  wings  must  be  observed,  as 
these  are  of  generic  importance  (fig.  101). 

The  wing  venation  of  the  Culicidce  is  comparatively 
simple.  Here  we  follow  closely  in  this,  as  in  other 
respects,  the  description  by  Mr.  Theobald.  It  is  an  easy 
one  to  work  with  (fig.  100). 

The  thickened  edge  is  called  the  costa,  it  forms  the 
free  edge  of  the  wing.     The  scales  on  it  are  of  no  generic 


Fig.  100. — Neuration  of  Wing  (Theobald). 


value  ;  these  may  differ  greatly  from  those  on  the  longi- 
tudinal veins.  The  scales  on  the  costa  in  all  genera  are 
mainly  lanceolate.  They  are  of  unequal  length,  arranged 
in  two  tiers  with,  at  their  bases,  a  third  row  arranged 
obliquely ;  these  last  are  more  like  the  scales  on  the 
longitudinal  veins.  The  straight  edge  of  the  wing,  with 
the  wing  expanded,  is  the  anterior  edge,  and  is  therefore 
so  described.  Next  to  the  costa  is  a  vein  running  from 
the  base  or  attachment  of  the  wing  to  rather  more  than 
half-way  to  the  tip,  terminating  in  the  costa;  this  is  called 
the  sub-costal  vein  (sc). 


2IO 


WING   SCALES 


I  2 

Anopheles.  Cyclolepptt  roil. 


Jcuithitiosoiiia. 


\J'<^        ■•, 


JMansonia. 


Stegomyia. 


Eretmapodiles. 


Psorophora. 


Fig.  ioi. — Various  Forms  of  Wing  Scales  (Theobald),  i,  Scales  on  veins 
and  on  cosla  in  Anopheles  ;  2,  scales  on  veins  in  Cyclolepptaon ;  3,  scales 
on  veins  and  on  costa  in  Janthinosottia;  4,  scales  on  veins  in  Mansonia; 
5,  scales  on  veins  in  Stegomyia ;  6,  scales  on  veins  in  Eretmapodites  ;  7,  scales 
on  veins  and  on  cosla  in  Cultx ;  8,  scales  on  veins  in  Mucidus',  9,  scales  on 
veins  and  on  costa  of  Psorophora. 


WING   VENATION  211 

The  other  veins  running  from  the  base  towards  the 
tip  are  known  by  numbers,  the  most  anterior  being  the 
first  longitudinal.  This  is  a  single  vein  running  the 
whole  length  of  the  wing  and  terminating  at  the  tip.  It 
is  covered  with  scales  in  its  whole  extent. 

The  second  longitudinal  arises  from  the  first  nearly 
half-way  from  the  base,  and  bifurcates  before  reaching 
the  tip.  The  space  enclosed  in  the  bifurcation  (D)  is 
known  as  the  first  fork-cell. 

The  third  longitudinal  arises  in  the  base  of  the  wing, 
but  is  not  covered  with  scales  for  nearly  the  first  two- 
thirds,  and  therefore  appears  merely  as  a  yellowish  line. 
It  does  not  bifurcate. 

The  fourth  longitudinal  arises  from  the  base,  is  covered 
with  scales  in  its  whole  extent,  and  bifurcates  near  the 
tip,  forming  the  second  fork-cell  (G). 

The  fifth  longitudinal  arises  at  the  base,  is  covered  with 
scales  in  its  whole  extent,  and  bifurcates  half-way  up  the 
wing,  enclosing  the  third  fork-cell  (K). 

The  sixth  does  not  bifurcate,  and  terminates  in  the 
costa  about  the  middle  of  the  posterior  border  of  the 
wing.  There  are  markings  or  thickenings  on  the  wing 
between  the  fifth  and  sixth  longitudinal  and  posterior 
to  the  sixth,  which  have  no  scales  and  are  not  regarded 
by  Mr.  Theobald  as  veins.  The  most  constant  of  these 
is  scaled  in  some  species  of  mosquito,  which  are  placed  by 
Theobald  in  a  separate  sub-family  Heptafthlebomyina.  Con- 
necting the  second  and  third  longitudinal  veins  is  the 
transverse  vein.  The  third  and  fourth  are  connected  by 
the  middle  transverse  vein,  and  from  the  fourth  longi- 
tudinal to  the  anterior  division  of  the  fifth  is  the  posterior 
transverse  vein,  They  are  definite  bands  of  considerable 
thickness  and  often  contain  air.  They  are  not  scaled. 
The  relative  positions  of  these  three  transverse  veins  is 
of  some  importance  in  the  separation  of  species.  Varia- 
tions cannot  be  relied  on  implicitly  for  this  purpose,  as 
in  some  species  the  arrangement  of  the  transverse  veins 
varies  considerably  in  different  individuals. 


212  classification 

Classification  of  Mosquitoes. 

In  a  work  of  this  kind  it  is  not  necessary  to  consider 
more  than  the  identification  of  the  commoner  and  more 
important  genera  into  which  the  Culicidce  are  divided. 

The  Culicidce,  as  has  been  indicated  in  the  previous 
chapter,  constitute  a  family  in  the  sub-order  Orthotrhapha 
nemocera  of  the  order  Dip/era.  Their  systematic  classi- 
fication has  been  attempted  by  several  writers,  but  that  of 
Mr.  Theobald,  which  is  based  largely  on  the  scale  char- 
acters of  the  mature  insect,  has  been  here,  in  the  main, 
adopted. 

The  main  characteristics  of  the  family  have  been 
already  given.  In  brief,  it  may  be  stated  that  the 
Culicidce  may  be  told  by  the  venation  of  the  wing  and 
by  the  arrangement  of  scales  on  the  head,  body  and 
veins  of  the  wing.  Other  insects  which  are  often  mis- 
taken for  these  have  a  different  form  of  wing  venation, 
and  the  wings  are  either  bare  or  ornamented  with  hairs. 

The  male  external  genitalia  are  by  some  considered 
to  be  of  great  importance  in  differentiation  of  genera. 
The  external  genitalia  vary  greatly  in  different  species. 
The  general  type  (see  fig.  98)  may  be  described  as  con- 
sisting of  two  large  fleshy  basal  lobes,  each  with  a  terminal 
chitinous  clasp  segment,  always  curved  and  often  orna- 
mented with  spines.  Between  the  claspers,  arising  in- 
ternally and  ventrally  to  the  claspers,  are  other  chitinous 
processes,  the  harpes,  which  may  be  well  developed,  formed 
of  two  segments,  or  rudimentary. 

Between  the  harpes  and  the  claspers  are  a  pair  of 
clasping  organs,  the  harpagones. 

The  chitinous  lobes  above  the  cloaca,  the  setaceous 
lobes,  are  part  of  the  rudimentary  eighth  segment. 

The  variations  of  each  part  are  great.  They  should  be 
examined  both  in  the  dried  specimen,  in  order  to  see  the 
relative  positions  of  the  various  parts,  and  after  treatment 
with  liquor  potassae,  or  in  flattened  specimens,  rendered 
transparent  in  order  to  make  out  the  details. 

The  family  Culicidce  is  divided  into  various  sub- 
families. 


SUB-FAMILIES 


213 


(1)  Sub-family  Corethrince* — Proboscis  short  and  not 
adapted  for  piercing,  palps  dependent.  These  insects 
are  incapable  of  biting  man  and  animals,  and  play  no 
part  in  the  transmission  of  disease  (fig.  102,  e). 

(2)  Sub-family  Megarhinince. — Large,  brilliantly  coloured 
insects.  Proboscis  long  and  bent  downwards.  Palps 
thin  towards  the  extremity  and  bent  upwards,  those  of 
the  male  being  composed  of  five  segments,  those  of  the 
female  varying  according  to  genera.  Head  clothed  with 
flat  scales  only  (fig.  102,  F,  and  fig.  97,  4). 

These  mosquitoes  are  found  as  a  rule  only  in  the 
vicinity  of  the  jungle.     They  rarely  attack  man. 

Larvae  are  larviverous.  They  have  a  very  short  respira- 
tory siphon. 

Head,  thorax  and  abdomen  thickly  covered  with  flat, 
square-ended  scales.  Scales  of  wings  small  and  square- 
ended.  First  sub-marginal  cell  much  shorter  than  second 
posterior.  Caudal  tuft  of  hairs  on  each  side  of  abdomen 
in  some  species. 

(3)  Sub-family  Anophclincv. — Proboscis  straight  and 
adapted  for  piercing.'  Palps  very  nearly  as  long  as  the 
proboscis  in  both  sexes,  composed  of  five  segments  in 
the  male  and  four  in  the  female  ;  the  last  two  joints  in 
the  male  are  expanded.  Antennas  plumose  in  the  male 
and  pilose  in  the  female. 

Head  has  numerous  upright  forked  scales,  and  a  few 
narrow  curved  scales  and  flat,  square-ended  scales  at  the 
sides  in  some  of  the  genera.  Thorax  and  abdomen 
ordinarily  have  few  scales  as  compared  with  other 
Culicidce.  Scutellum  simple,  never  trilobed.  The  varied 
covering  of  thorax  and  abdomen  is  the  basis  of  the  sub- 
division into  genera.  Wings  in  most  genera  marked  with 
black  or  brown  patches  ;  wing  scales  long  and  lanceolate 
or  fusiform. 

The  female  has  only  one  spermatheca.     The  eggs  are 


*  In  Theobald's  latest   classification  the  Corethrince   are  not  con- 
sidered as  belonging  to  the  Culicidce. 


SUB-FAMILIES 


Fig.  102. 


A — Lateral  view  of  Anopheline. 

B — Lateral  view  of  Culicine. 

C — Anopheline  viewed  from  above. 


D — Culicine  viewed  from  above. 
E  —  Head  of  Corethra. 
F — Head  of  Megharinina. 


Fig.    103.  — i,  Culicine  male;    2,  Culicine  female;    3,  Anophelina  male 
4,  Anophelina  female. 


2l6  SUB-FAMILIES 

laid  singly,  are  more  or  less  boat-shaped  and  float  on 
the  water,  owing  to  the  presence  of  lateral  air  chambers. 
Larvae  have  no  respiratory  siphon,  and  when  at  rest  lie 
horizontally  on  the  surface  of  the  water.  Pupa?  have 
trumpet-shaped  respiratory  siphons. 

The  adult  mosquito  appears  very  narrow  when  viewed 
from  above,  and  as  seen  in  profile  the  long  axes  of  the 
proboscis,  thorax,  and  abdomen  form  an  almost  straight 
line.  When  at  rest  on  a  flat  surface,  the  insect  commonly 
presents  an  appearance  as  if  standing  on  its  head. 

(4)  Sub-family  Culicince. — Palps  in  the  3  as  long  or 
longer  than  the  proboscis  ;  in  the  2  always  much  shorter 
than  the  proboscis  ;  metanotum  nude,  scutellum  trilobed. 

Viewed  from  above,  these  mosquitoes  appear  much 
broader  than  Anophclincv,  and  from  a  lateral  aspect  have 
a  hunchbacked  appearance,  which  is  very  different  from 
that  of  the  Anopliclince  (fig.  102). 

The  eggs  differ  markedly  in  different  genera  of  the 
Culicines,  both  in  shape  and  the  manner  in  which  they 
are  laid ;  in  the  genus  Stegomyia  and  others  they  are  laid 
singly,  but  never  resemble  those  of  Anophelina:,  while  in 
Cidex  and  others  they  are  laid  in  rafts. 

The  larvae  are  provided  with  a  respiratory  siphon,  and 
when  at  rest  lie  obliquely  in  the  water  with  the  head 
downwards. 

(5)  Sub-family  /Ed'unv. — Many  of  the  genera  formerly 
included  under  this  sub-family  are  now  separated  from 
it,  and  Theobald  makes  several  new  sub-families.  These 
are  :  JEdince}  Limatincv,  Deinoceratince,  Uranotcenincc,  Den- 
dromyincB.  Palps  very  short  in  both  sexes.  Antenna.'  in 
the  males  not  always  plumose.  The  proboscis  may  be 
very  long,  and  is  sometimes  clubbed  at  the  extremity. 
Metanotum  nude,  without  hairs  or  scales  in  most,  but 
in  the  sub-division  Dendromyince  there  is  always  a  tuft 
of  hairs  on  the  metanotum. 

This  sub-family  has  been  less  studied  than  Anophelina 
or  Culicina.  Most  of  its  members  are  jungle,  mangrove 
or  forest  mosquitoes.     Many  of  them  bite  by  day.     They 


SUB-FAMILIES  217 

breed  in  natural  collections  of  water,  often  in  the  hollow 
axils  of  leaves,  in  pitcher  plants  or  in  perforated  bamboos. 
They  may  also  breed  in  swamps,  slowly  moving  water, 
roadside  trenches,  or  in  streams. 

The  remaining  sub-families  are  represented  by  a  few 
rare  species  and  are  of  little  importance  to  medical 
workers. 

(6)  Sub-family  Joblotiiuv  (Trichoprosoponince,  Theobald). 
Head,  thorax  and  abdomen  covered  with  square-ended 
scales.  Palps  short  in  both  sexes.  Metanotum  adorned 
with  hairs  and  square-ended  scales. 

(7)  Sub-family  Heptaphlebomyincv.  Head,  thorax,  scu- 
tellum,  metanotum  and  abdomen  as  in  Culex.  Wings 
have  a  seventh  scaled  longitudinal  vein. 

Four  species  known.  H.  simplex  (Theobald,  1903)  from 
Central  Africa  (Mashonaland)  was  the  first  described. 

Theobald's  latest  grouping  of  the  Culicidce  is  modified 
from  that  of  Lutz,  and  differs  in  some  respects.  He 
divides  the  Culicidce  as  follows  : — 

Culicidce 


Anophelince.  Orthorrhynchce.  Megarhinincc. 

Proboscis  straight,  palpi  Proboscis  straight,  palpi  short         Proboscis  curved,  palpi  short 

long  in   ?  and  S  in   5  ,  long  or  short  in  g  or  long  in  J  ,  long  in  S 


Metanotopsilce  Melanotolriclne 


Heteropalpa.  Micropalpa.  Heleropalpa.  Micropalpa. 

Palpi  short  in    5  ,  long  Palpi  short  in  J         Palpi  short  in  J,        Palpi  short  in   J 

in  $  and  $  long  in  $  and  $ 


I                              I.I  I  I  I.I 

"uhtiiicc.  Heptaphlebomyune.     ALduiiv.  U.ranotcenina.  7'richopro-   Dendromynne.   Limatune. 

Wings,  6              Wings  with          First  fork-  First  fork-  soponina:.  Proboscis         Proboscis 

ngitudinal          7  longitudinal        cell  large  cell  large  straight.  elbowed, 
aled  veins           scaled  veins 

There  are  many  points  in  favour  of  this  classification. 

(1)  Corethrince  are  separated  off  from  the  Culicidce  and 
considered  as  a  distinct  family,  the  Corethridce. 


2l8  CLASSIFICATION 

(2)  AnophelincB  and  Megarhinince,  two  groups  which 
differ  at  all  stages  of  their  development  from  the  rest  of 
the  Culicidce,  are  separated  off  from  the  other  groups. 

(3)  The  old  group  ALdeomyince,  containing  several 
markedly  dissimilar  sub-groups,  is  broken  up  so  that 
the  JEdince,  Uranotcenince,  Dendromyince,  Limatince  and 
Deinoceratinince,  which  are  naturally  distinct  groups,  are 
now  widely  separated. 

Though  we  consider  that  the  scheme  is  one  which  will 
be  of  practical  value  and  a  useful  aid  in  the  subdivision 
of  the  Culicidce,  we  have  not  thought  it  advisable  to  adopt 
it  in  the  present  unsettled  condition  of  the  classification 
of  these  insects,  as  it  has  not  received  the  general  approval 
of  those  working  with  mosquitoes. 

(1)  Genera  of  sub-family  Corethrince. 

Metatarsus  longer  than  first  tarsal  joint Corethra. 

Metatarsus  shorter  than  first  tarsal  joint  Mochlcmyx. 

(2)  Genera  of  sub-family  Megarhiniucu.  This  sub- 
family is  divided  by  Theobald  into  three  genera. 

A.  Palpi  long  in  both  sexes. 

(a)  Last  segment  of  ?  palpi  round  or  blunt  as  if 

broken Megarhinus . 

{b)  Last  segment  of  ?  palpi  long  and  pointed...     Ankylorhynchus- 

B.  Palpi  of  ?  short. 

Palpi  not  more  than  one-third  length  of  the 

proboscis   ToxorhynchUes. 

The  differences  between  these  three  genera  are  com- 
paratively slight,  and  a  division  founded  on  the  absence 
or  presence  of  lateral  caudal  tufts  would  be  simpler,  but 
would  not  quite  correspond  with  Theobald's  division. 

(3)  Genera  of  sub-family  Anophdincv  (Theobald). 
Theobald  now  subdivides  this  sub-family  into  21  genera. 

The  practical  value  of  the  subdivision  of  the  Anopheliiuv 
into  so  many  genera  is  disputed  by  many  medical  men. 
There  are  few  branches  of  natural  history  in  which  such 
enormous  advances  have  been  made  as  in  our  knowledge 
of  the  Culicidce,  and  each  year  there  are  additions  to  this 
knowledge. 


ANOPHELIN.E  210 

Of  the  Anophelince  about  130  species  are  now  known. 
This  is  too  large  a  number  to  be  conveniently  grouped 
into  one  genus.  A  subdivision  is  necessary,  and  some 
grouping  will  be  made  automatically  by  any  one  working 
with  these  insects.  It  is  better  that  a  uniform  system 
should  be  adopted  than  that  each  worker  should  make 
his  own  groups. 

The  subject  is  so  large  that  it  is  now  a  special  branch, 
and  we  propose  merely  to  deal  with  selected  parts  of  it 
and  select  such  groups  as  are  represented  by  common 
species,  and  by  species  known  to  carry  malaria. 

It  is  unfortunate  that  the  grouping  into  genera  based 
on  the  external  characters  of  the  adults  does  not  corre- 
spond with  grouping  according  to  the  power  of  carrying 
malaria,  or  with  the  class  of  breeding  place  or  the  habits 
of  the  mosquitoes.  In  one  genus,  Myzomyia,  are  included 
the  harmless  M .  rossi  and  the  harmful  M.  funesta,  the 
first  essentially  a  foul  or  stagnant  water  breeder,  the 
second  breeding  in  fresh  water,  preferably  in  streams. 

In  another  genus,  Anopheles,  are  included  A.  macnli- 
peiinis,  which  hibernates  through  an  English  winter, 
whilst  the  larvae  are  killed  by  cold,  and  A.  bifurcatus, 
which  is  killed  by  cold,  though  the  larvae  can  survive 
an  English  winter. 

Anophelince. 

Of  the  21  genera,  ten  are  represented  by  one  species 
only ;  one,  Stethomyia,  is  represented  by  four  species,  in- 
cluding Stethomyia  jragilis  (Anopheles  treacherii).  This 
genus  is  very  similar  to  Anopheles,  in  that  the  scales  on  the 
wings  are  all  of  the  same  colour  and  that  the  mosquitoes 
have  a  peculiarly  "  bald"  appearance.  The  flat  scales  on 
the  head  on  which  the  separation  is  now  based  are  very 
scanty.  The  mammilation  of  the  prothoracic  lobes,  which 
was  formerly  described  as  the  characteristic,  is  not  present 
in  all  species.  There  seems  little  good  reason  for  separat- 
ing this  group  from  Anopheles. 

The  ten  genera,  represented  each  by  a  single  species, 


220  GENERA   OF   ANOPHELINjE 

and  in  some  cases  by  a  single  specimen,  are  separated  off 
on  various  grounds.     They  arc  : — 

Feltinella,  in  which  the  basal  lobe  of  the  male  genitalia 
is  divided  into  two  segments.    One  species,  F.  pallidapalpi. 

Chrystya,  similar  to  Myzorhynchus,  but  separated  by  the 
possession  of  long  lateral  tufts  of  hairlike  scales  on  the 
abdomen. 

Lophoscelomyia,  resembles  Nyssorhynchus,  but  differs 
in  that  there  are  long  tufts  of  scales  on  the  femora  of  the 
hind  legs.     Bamboo  breeder. 

Kerteszia.  —  Intermediate  between  Myzorhynchus  and 
Cell  ia. 

Bironella. — Male  only  known. 

Chagasia. — Antennae  of  female  have  whorls  of  scales 
as  well  as  hairs,  and  dense  outstanding  scales  at  the  side 
of  the  thorax. 

Aldrichia. — Thorax  and  abdomen  scaled  as  in  Culex. 
One  specimen. 

Neomyzomyia. — Is  allied  to  Myzomyia  and  Pyretophorust 
but  is  at  once  told  by  the  dense  tufts  of  scales  at  the 
posterior  angles  of  the  head  and  on  the  prothoracic 
lobes.     One  species  known,  X.  elegans. 

Calvertina. — Closely  allied  to  Chagasia.  Antenna  with 
outstanding  scales  on  the  second  segment,  more  appressed 
ones  on  the  first.  At  least  one  segment  of  the  abdomen 
with  long,  flat,  more  or  less  spatulate  scales.  One  specimen 
known. 

Manguinhosia. — Thorax  with  narrow  hair-like  curved 
scales,  and  some  of  them  broad  straight  scales,  others 
spatulate  on  the  sides.  Abdomen  covered  with  line  hairs, 
except  the  three  last  segments,  which  are  covered  with 
scales.  Tufts  of  scales  on  hind  femora.  Wing  scales 
lanceolate. 

The  more  important  of  the  groups  or  genera  of  Anophe- 
liiiiv  are  Anopheles,  Myzomyia,  Cycloleppteron,  Stethomyia, 
Pyretophorus,  Myzorhynchus,  Nyssorhynchus,  Cell  id. 

i.   Thorax  and  abdomen  with  hair-like  curved  scales. 
(a)  Only  upright  fork  scales  on  head,  wing  scales 

lanceolate  and  uniform  in  colour    Anopheles. 


GENERA   OF   AXOPHELIN^E  221 

(b)  Some  flat  scales  as  well  as  upright  fork  on 

head.     Otherwise  like  Anopheles  Stetliomyia. 

(c)  Only  upright  fork  scales  on  head.  Scales  on 
wings  mostly  long    and   narrow  and  of  two 

colours     Myzomyia. 

(d)  Only  upright  fork  scales  on  head.  Scales  on 
wings  partly  large  and  inflated.     Wing  scales 

of  two  colours    Cycloleppteron. 

2.  Thorax  with  distinct  narrow  curved  scales  :  abdo- 
men hairy.     Wing  scales  of  two  colours. 

(a)  Wing  scales  small  and  lanceolate  :  head  with 

ordinary  upright  fork  scales    Pyretophorus. 

(6)  Wing  scales  broad,  lanceolate :  head  with 
broad  scales,  not  closely  appressed,  but  not 
forked  or  fimbriated Myzorhynchella. 

3.  Thorax  with  hair  like  curved  scales.  Wing 
scales  of  two  colours.  Scales  on  head  upright 
fork. 

(a)  Some  narrow  curved  scales  in  front  of  thorax  : 
abdomen  with  apical  lateral  scale  tufts  and 
scaly  venter,  no  ventral  tuft    Arribalzagia. 

(/>)  Abdominal  scales  on  venter  only,  no  lateral 
abdominal  tufts,  but  a  distinct  ventral  apical 

tuft.     Palps  in   ?   densely  scaled   Myzorhynchus 

(4)  Thorax  and  abdomen  with  scales.    Wing  scales 

of  two  colours.     Upright  fork  scales  on  head. 

(a)  Thoracic  scales  narrow  curved  to  spindle- 
shaped  :  abdominal  scales  as  lateral  tufts  and 
small  dorsal  patches  of  flat  scales Nyssorhy?ichus. 

(ft)  Abdomen    nearly    completely   covered    with 

irregular  scales  and  lateral  tufts Cellia. 

(c)  Similar  to  Cellia  but  no  lateral  scale  tufts    ...     Neocellia. 

Some  authorities  object  to  such  subdivision  and  it  has 
been  urged  that  as  in  any  one  country  the  number  is 
comparatively  small  a  complete  description  of  the  species 
found  in  each  country  would  suffice  for  practical  pur- 
poses, but  with  this  argument  we  do  not  agree. 

On  the  other  hand,  some  of  Theobald's  points  are  very 
difficult  to  make  out,  and  this  is  particularly  so  as  regards 
the  wing  scales.  Some  groups  are  easily  separated,  and 
between  Anopheles  on  the  one  hand  and  Cellia  on  the 
other  the  differences  are  very  marked.  A  division  into 
four  to  six  genera  would  probably  suffice. 


222  SPECIES    OF    AXOl'liKI.IX.K 

Genus  Anopheles. — Thorax  and  abdomen  clothed  with 
hairs  only;  the  palpi  in  the  female  are  thin,  not  densely 
scaled,  and  generally  unhanded.  Wing  veins  covered 
with  long  lanceolate  scales,  which  may  or  may  not  form 
spots.  These  spots,  if  present,  are  never  so  numerous  a^ 
in  other  genera,  and  are  not  formed  by  different  colouring 
of  the  scales.  In  the  female  a  single  spermatheca  only 
is  present. 

These  mosquitoes  may  be  said  to  be  characterized  by  then- 
extreme  baldness.     They  are  of  comparatively  large  size. 

The  genus  includes  two  well-known  carriers  of  the 
parasites  of  malaria  in  sub-tropical  and  temperate  climates, 
A.  maculipennis  and  A.  bifurcatus. 

Anopheles  maculipennis,  the  type  of  the  genus,  is  easily 
recognized.  It  is  a  yellowish-brown  mosquito ;  neither 
legs,  proboscis,  nor  palps  are  banded.  P'our  black  spots 
on  the  wing  formed  by  accumulation  of  scales.  It  does 
not  assume  the  Anopheline  position  as  markedly  as  most 
Anophelines. 

This  mosquito  is  widely  distributed  in  Europe,  from 
the  borders  of  the  Mediterranean  to  Scandinavia.  It  is 
found  also  in  Algeria,  Palestine,  the  United  States  and 
Canada.  In  England  it  is  common  ;  according  to 
Nuttall  its  distribution  in  England  agrees  to  some  extent 
with  the  old  malarious  districts. 

A.  maculipennis  is  the  most  active  propagator  of  malaria 
in  Europe,  Algeria,  Tunis,  and  the  United  States.  It  has 
been  shown  to  be  capable  of  serving  as  a  definitive  host 
for  all  forms  of  the  malaria  parasite.  It  is  also  an  inter- 
mediate host  of  Filaria  immitis. 

In  the  winter  in  temperate  climates  the  larvae  die,  but 
the  imago  can  hibernate  all  through  an  English  winter. 

Anopheles  bifurcatus  is  easily  distinguished  from  the 
preceding  species  by  having  no  spots  on  the  wings. 
Its  distribution  corresponds  in  a  general  way  with  that 
of  A.  maculipennis,  but  is  a  less  common  species  and  does 
not  frequent  houses. 

This  mosquito  is  also  an  active  carrier  of  malaria,  and 


SPECIES   OF   ANOPHELIN^E  223 

has  been  shown  to  be  more  easily  infected  experimentally 
than  even  A.  maculvpennis.  In  an  English  winter  the  adult 
forms  are  killed,  but  the  larvae  remain  alive  even  if  the 
water  be  frozen  throughout  the  winter.  Stethomyia,  with  a 
limited  number  of  genera,  would  be  included  in  Anopheles. 

Genus  Myzomyia. — Thorax  and  abdomen  with  hair- 
like curved  scales.  The  wings  are  spotted,  and  have 
mostly  long,  thin,  or  narrow  lanceolate  lateral  vein  scales. 
They  are  usually  small  or  moderate-sized  mosquitoes. 
Head  has  only  a  few  upright  forked  scales. 

Myzomyia  rossii  is  distinguished  by  the  ornamentation 
of  the  wings.  Along  the  costa  are  four  large  patches  of 
dark  brown  or  black  scales.  The  large  middle  spot  has 
a  small  dark  spot  below  it  in  the  centre,  giving  it  a  T- 
shaped  appearance. 

This  mosquito  is  the  "  large  dappled-winged  mosquito  " 
which  Major  Ross,  in  his  work  in  1899,  in  Calcutta,  failed 
to  infect  with  malaria.  It  is  a  common  mosquito  in 
various  parts  of  India  and  the  East,  chiefly  in  the  neigh- 
bourhood of  towns.  It  breeds  in  muddy  pools  or 
shallow  tanks,  even  in  cesspools. 

This  mosquito  has  never  been  found  infected  with  the 
parasites  of  human  malaria  in  nature.  It  may  serve  as  an 
intermediate  host  for  Filaria  bancrofti. 

Myzomyia  funesta. — A  small,  rather  dark  mosquito.  The 
black  costa  is  marked  by  six  pale  spots ;  there  is  always 
a  pale  costal  spot  near  the  base.  The  black  scales  upon 
the  wing  veins  are  also  interrupted  by  white  spots.  There 
are  pale  spots  on  the  fringe  of  the  wings  at  the  points  of 
insertion  of  the  longitudinal  veins. 

This  mosquito  is  widely  distributed  in  Central  and 
West  Africa,  and  is  an  important  carrier  of  the  parasites 
of  human  malaria  in  those  regions.  It  frequents  houses, 
but  does  not  leave  them  in  the  daytime,  hiding  in  dark 
corners  high  up  out  of  the  reach  of  the  breeze  from  doors 
and  windows.  It  feeds  in  the  early  hours  of  the  evening 
by  preference,  but  at  other  times  as  well. 

Myzomyia    culicificacies. — Has  unbanded  leg's,  and  the 


224  SPECIES   OK   AXOPHELIXiK 

largest  light  costal  area  near  the  base  of  the  wing.  There 
are  only  three  light  ureas  on  the  fringe  of  the  wings.  It 
appears  to  be  a  carrier  of  malaria  in  India.  It  assumes 
the  position  of  a  culex  when  at  rest. 

Pyretophorus. — Thorax  with  narrow  curved  scales,  not 
hairlike  as  in  Myzomyia.  Abdomen  with  hairs,  and  a  few 
scales  on  the  genital  lobes.  Wings  with  small,  short, 
lanceolate  or  narrowish  scales,  much  spotted  as  a  rule. 
Legs  banded,  sometimes  spotted.  The  differences  from 
Myzomyia  are  not  obvious. 

Pyretophorus  costalis. — Told  by  the  curious  mottled 
character  of  the  femora  and  tibiae.  The  wings  may  be 
said  to  be  white  spotted  with  black,  in  contrast  to  .1/. 
funesta,  which  are  black  spotted  with  white. 

This  species  is  also  widely  distributed  over  the  whole 
of  Central  Africa  and  the  West  Coast.  The  larvae  are 
found  in  abundance  during  the  rainy  season  in  puddles  in 
West  Coast  towns.  It  is  the  most  common  Anopheline 
on  the  sea-coast. 

Like  A.  maculipennis  it  serves  as  a  definitive  host  for 
the  parasites  of  all  forms  of  human  malaria.  It  also  is  an 
occasional  intermediate  host  of  F.  bancrofti. 

Genus  Myzorhynchus. — Thorax  with  hair-like  scales. 
Abdomen  with  ventral  scales  and  a  ventral  apical  tuft  of 
black  scales.  Wing  scales  moderately  broad  and  lanceo- 
late. Palpi  and  proboscis  densely  scaled.  In  some 
species  this  scaling  is  so  thick  that  the  proboscis  and  palps 
appear  to  be  very  thick  as  seen  with  the  naked  eye. 

Myzorhynchus  sinensis. — A  brownish  mosquito.  Thorax, 
slaty-grey  background  with  purplish-brown  longitudinal 
stripes,  adorned  more  or  less  with  pale  golden  scales. 
Found  in  China,  Formosa,  Malay  Archipelago,  &c. 

Myzorhynchus  barbirostris. — In  this  species  the  palpi  are 
densely  covered  with  deep  black  scales.  The  prothoracic 
lobes  have  dense  tufts  of  large  black  scales  projecting 
forwards.     Found  in  Malaya  and  Old  Calabar. 

Both  these  species  can  carry  filaria.  They  are  difficult 
to  infect  with  parasites  of  malaria,  and  are  probably  not 


CULICINA  225 

important  carriers,  as  they  may  be    numerous  in  towns 
where  malaria  is  not  prevalent. 

Genus  Nyssorhynchus. — Thorax  with  narrow  curved  and 
spindle-shaped  scales.  Abdomen  with  flat  ventral  scales, 
and  sometimes  latero-dorsal  patches.  Wing  scales  bluntly 
lanceolate.  (The  legs  are  always  banded  or  spotted  with 
white). 

N.  fuligiiiosus. — Probably  the  "  small  dapple-winged 
mosquito"  of  Ross. 

Genus  Cellia. — Thorax  with  flat  spindle-shaped  scales. 
Abdomen  scaled,  the  scales  irregularly  disposed  on  the 
dorsum  and  on  the  venter.  Two  dense  bifid  tufts  on  the 
ventral  aspect  of  each  segment.  Palpi  densely  scaled. 
Wings  covered  with  large  bluntly  lanceolate  scales. 

Cellia  pharcensis  is  found  in  Africa,  C.  kochii  in  Asia, 
C.  argyrotarsis  and  C.  albimana  in  the  West  Indies  and 
South  America. 

These  mosquitoes  may  breed  in  fairly  dirty  water. 
C.  kochii  is  commonly  found  in  outlying  villages  or 
suburbs  of  towns.     They  are  night  biters  as  a  rule. 

C.  argyrotarsis  is  one  of  the  carriers  of  malaria  in 
Tropical  America,  but  according  to  Darling,  C.  albimana 
is  of  more  importance.  It  differs  from  C.  argyrotarsis  in 
that  the  last  tarsal  joint  of  the  hind  legs  is  not  completely 
white.  C.  kochii  probably  acts  as  a  carrier  in  the  Malay 
Peninsula  and  Archipelago.  These  mosquitoes  are 
common  in  the  most  malarial  settlements  in  these 
countries. 

CULICINA. 

Genera  of  the  sub-family  Culicina. — This  sub-family  in- 
cludes a  large  number  of  mosquitoes.  Numerous  genera* 
have  been  created  by  Mr.  Theobald  with  a  view  to 
simplifying  the  identification.  It  is  not  proposed  to  deal 
here  with  all  of  these  genera,  but  only  such  as  are  known 
to  be  of  importance  from  the  medical  point  of  view- 
A  table  is   appended,    but   those  who    wish    to    go    into 

*  Theobald  now  makes  eighty-three  genera  of  Culicina. 
15 


226  CULICINA 

the     matter     more     fully     should     consult     Theobald's 
monograph. 

The  type,  Culex  pipiens,  has  narrow  curved  scales  on  the 
head  and  scutellum.  On  the  head  there  are  also  upright 
fork-scales  at  the  back,  and  flat  tile-shaped  scales  at  the 
sides  (see  diagram,  p.  210).  The  lateral  scales  on  the 
veins  of  the  wings  are  long  and  narrow,  those  running 
along  the  vein  are  shorter  and  broader. 

1.  Certain  genera  can  be  told  at  once  by  the  char- 
acters of  the  scales  on  the  wing,  though  in  other 
respects  they  may  resemble  the  genus  Culex: 
{a)  Wing  scales  broad   and  asymmetrical  (see 

fig.  101,  p.  210)    Mansonia. 

(b)  Wing  scales  broad-ended  or  pyriform,  sym- 
metrical, and  often  parti-coloured  (see  fig.  101, 
p.  210) Mucidus. 

{c)  Wing  scales  thick  and  elongated,  ending 
either  diagonally  or  convexly,  more  or  less 
bluntly  pointed Tantorhynchus. 

2.  Other  important  genera  are  characterised  by 
having  the  head  and  scutellum  entirely  covered 
with  flat,  square-ended  scales  arranged  like 
tiles  on  a  roof  (see  fig.  97,  1). 

(a)  Palpi  of   ?    short,  of  3  thickened  apically 

and  tufted Stegomyia. 

(b)  Palpi  of   2  longer  than  in  Stegomyia,  of  3 

long,  thin,  acuminate  and  without  tufts  Desvoidea. 

3.  Other  genera  have  head  and  scutellar  scales  of 
the  Culex  type,  but  are  differentiated  on  various 
grounds. 

(a)  Wing  scales  long,  narrowly  lanceolate,  and 

collected  in  spots  ;  palpi  clubbed  in  the   3      Theobaldia. 

(b)  Wing  scales  at  apex  of  veins  dense  and 
rather  broad  ;  femora  swollen.  Small  dark 
mosquitoes    Melanconio?i. 

{c)  Wings  with  short,  thick,  median  scales, 
and  short,  broad  lateral  ones  on  some  of 
the  veins  ;  scales  mottled GrabJiamia. 

4.  Peculiarlv  twisted  scales  arranged  in  whorls  on 
the  sixth,  seventh,  eighth,  and  sometimes  ninth 
joints  of  the  antennae  in  the  males.  The 
females  show  no  similar  scaling.  Fairly  com- 
mon   mosquitoes    throughout    Malaya    and   in 

Brazil    Lophocei-atomyia. 


CULICINA  227 

For  purely  diagnostic  purposes  Theobald  arranges  the 
genera  somewhat  differently.  The  table  subjoined  is 
based  on  this  arrangement. 

A.  Legs  more  or  less  densely  scaled. 

(a)  Head  not  entirely  clothed  with  flat  scales. 

1.  All  the  legs  densely  scaled. 

Wings  with  large  pyriform  scales  ...  Mucidus. 

Wings  with  narrow  scales  Psorophora. 

2.  Hind  legs  only  densely  scaled    Janthinosoma. 

B.  Legs  uniformly  scaled  with  flat  scales. 

(a)  Head   and   scutellar  scales   all   flat   and 
broad. 

1.  Palpi  of  ?     short,  of  g  thickened 

apically  and  tufted    Stegomyia. 

2.  Palpi  of  J  longer  than  in  Stegomyia, 

and  in  $  thin,  acuminate,  simple...     Desvoidea. 
{by  Head  scales  mostly  flat,  but  a  median  line 

of  narrow   curved  ones  ;  scutellar  scales 

flat  on  mid-lobe,  narrow  curved  on  lateral 

lobes;   $  palpi  longer  than  proboscis    ...     Macleaya. 
(c)  Head  scales  mostly  flat,  irregular,  narrow 

curved  ones  behind ;  mid-lobe  scutellum 

with  flat  scales,  lateral  lobes  with  narrow 

curved;   g    palpi  shorter  than  proboscis     Catageiomyia. 
{d    Head  scales  mostly  flat,  but  a  few  narrow 

curved  ones  in  middle  in  front  ;  scutellar 

scales  all  flat    Scutomyia. 

(e)    Head  with    flat   scales,    except   a    small 

median    area   of    narrow    curved    ones  ; 

scutellar  scales  all  narrow  curved   Howardina. 

{f    Head  with  all  flat  scales,  except  a  thin 

line     of    narrow    curved     ones   behind  ; 

scutellar  scales  all  narrow  curved  Danielsia. 

(^•)|Head  with  small  flat  scales,  over  most  of 

surface,  with  median  line  and  line  around 

eyes  of  narrow    curved   ones  ;    scutellar 

scales  bluntly  spindle  or  club-shaped Hulecaleomyia. 

'li)  Head  and  scutellar  scales  narrow  curved. 

1.  Wing  scales  long,  narrowly  lanceo- 
late, collected  in  spots;  palpi  clubbed 

in  g}  five-jointed  and  rather  long  in  ?      Theobaldia. 

2.  Wing  scales  (lateral)  long  and  nar- 
row ;    palpi  in    $    not  clubbed,   or 

hairy,  in  2  three-jointed    Culex. 


225  CULEX 

3.  Wing  scales  at  apex  of  veins  dense 
and  rather  broad  ;  femora  swollen. 

Small  dark  species  Melanoconion. 

4.  Wings  with  short,  thick  median  scales 
and  short,  broadish  lateral  ones  on 
some  of  the  veins  ;  scales  mottled  ; 

fork-cells  rather  short Grabhamia. 

5.  Wings  with  dense,  broadish,  elon- 
gated, truncated  scales    Tfeniorhynchus. 

6.  Wings  with  broad,  short,  asymme- 
trical scales    Mansonia. 

(z)  Head  covered  with  rather  broad,  flat, 
spindle-shaped  scales  ;  scutellum  with 
small  flat-scales  to  mid-lobe Gilesia. 

(/)  Head  clothed  with  flat,  irregularly  dis- 
posed scales  all  over,  with  patches  of 
narrow  curved  ones  ;    3  palpi  clubbed  ...     Acariomyia. 

{k)  Abdomen  with  projecting,  flat  lateral 
scales  with  deeply  dentate  apices  ;  wings 
not  ornamented    Lasioconops. 

(/)  Wings  ornamented;    scutellum  with   flat 

and  narrow  curved  scales  Finlaya. 

(m)  Head  flattened  laterally.  Palps  in 
female  nearly  half  the  length  of  the  pro- 
boscis. Spindle-shaped  scales  round  the 
eyes,  otherwise  scaled  as  in  Stegomyia. 
Common  jungle  mosquitoes  in  Malaya   ...     Leicestcria. 

Genus  Culex. — Head  with  narrow  curved  and  upright 
fork-scales  only  on  the  occiput  and  flat  tile-like  scales  at 
the  sides.  Narrow  curved  or  spindle-shaped  scales  on 
the  scutellum.  On  the  wing  veins  short  truncated  median 
scales  and  long,  thin  lateral  ones. 

The  genus  Culex  has  been  of  late  much  subdivided, 
but  is  still  a  very  difficult  one  and  includes  a  large 
number  of  species  that  rather  closely  resemble  each 
other. 

The  type  mosquito  of  the  genus  Culex  pipiens  is  a 
common  English  variety  and  is  widely  distributed  in  the 
temperate  regions,  and  may  be  met  with  in  houses  at  any 
time  of  the  year.  The  females  hibernate  in  cellars  and 
outhouses. 


STEGOMYIA  229 

Culcx  fatigaus. — Abdomen  dusky  black  with  basal  pure 
•white  bands  and  basal  white  lateral  spots ;  pleurae  and 
metanotum  chestnut-brown,  thorax  with  two  dark  parallel 
lines. 

This  mosquito  is  found  everywhere  in  tropical  and 
sub-tropical  countries.  It  is  a  domestic  species  and 
passes  its  life  in  the  vicinity  of  houses. 

It  is  the  chief  carrier  of  Filarla  nocturna,  and  also 
serves  as  the  definitive  host  of  Proteosoma  of  birds. 

Genus  Stegomyia. — For  the  most  part  black  and  white 
mosquitoes.  Head  completely  covered  with  broad,  flat 
scales,  some  upright  forked  scales  (fig.  97).  Mesothorax 
with  narrow  curved  or  spindle-shaped  scales.  Scutellum 
always  with  broad  flat  scales  on  the  middle  lobe,  and 
usually  with  similar  scales  on  the  lateral  lobes.  Abdomen 
completely  covered  with  flat  scales,  banded  or  unbanded, 
with  white  spots  on  the  lateral  aspect.  Wings  similar  to 
Culex ;  fork-cells  shorter.  Eggs  laid  singly.  Larvae  with 
short,  broad  respiratory  siphon. 

Mosquitoes  of  this  genus  have  a  wide  distribution  in 
the  tropical  zone.  They  are  all  hardy  mosquitoes, 
Stegomyia  calopus  (S.  fasciata)  being  especially  so. 

Stegomyia  calopus  (S.  fasciata)  is  distinguished  by  the 
marking  on  the  thorax,  which  has  a  curved  silvery  line  on 
each  side,  and  two  dull  yellow  narrow  parallel  ones  in  the 
middle.  This  marking  is,  however,  subject  to  some 
variation. 

This  species  is  widely  distributed  in  the  Tropics,  and  is 
also  found  in  temperate  climates,  owing  probably  to  the 
ease  with  which  it  may  be  carried  in  ships  as  ova,  larvae, 
or  adults.  It  is  the  carrier  of  Yellow  Fever  and  therefore 
most  important. 

Stegomyia  scutellaris  is  another  member  of  this  genus 
which  is  very  common  in  some  districts  :  it  is  important 
"to  distinguish  it  from  S.  calopus.  It  is  easily  recognized 
by  the  presence  of  a  single  broad  white  band  down  the 
centre  of  the  thorax.  Abdomen  and  legs  banded  black 
and  white  in  both  species. 


230  MANSONIA 

This  species  is  widely  distributed  in  Asia  and  is  a 
severe  biter.  It  is  common  in  the  jungle  and  may  be 
found  where  there  are  no  human  habitations,  but  is  also 
very  common  in  small  and  large  settlements,  and  may 
infest  houses  and  breed  in  similar  places  to  S.fasciata. 

Genus  Mansonia. — Head  clothed  with  narrow  curved 
scales  and  numerous  long  upright  forked  scales.  Thorax 
and  scutellum  with  narrow  curved  scales  and  many  hairs. 
Wings  densely  scaled  with  characteristic  broad,  asym- 
metrical scales  (fig.  101,  4).  Two  spermathecse  in  the 
female.  Eggs  are  in  the  form  of  a  bottle  with  an 
elongated  neck. 

This  genus  includes  several  species  commonly  found 
in  the  Tropics.  No  representative  of  the  genus  has  yet 
been  found  in  Europe. 

The  wing  scales  are  characteristic,  but  in  the  genus 
yEdeoniyia  scales  of  very  similar  appearance  are  found. 
It  is  of  importance,  therefore,  that  the  sub-family  should 
be  accurately  determined,  and  in  case  of  doubt  both  male 
and  female  should  be  examined. 

Members  of  this  genus  are  found  in  the  neighbour- 
hood of  marshes  and  along  the  course  of  rivers  and 
streams  with  sedge-grown  banks  and  edges. 

One  species,  M.  uniformis,  a  brownish  variety,  is  the 
common  carrier  of  filariasis  on  the  Zambesi  and  in  parts 
of  Central  Africa.     M.  annulipes  also  can  carry  filaria. 

M.  annulipes  is  a  banded  black  and  white  mosquito 
having  a  superficial  resemblance  to  a  Stegomyia.  Exam- 
ination with  a  hand  lens  will  show  the  characteristic 
marking  of  the  thorax,  three  whitish  spots  on  the  front 
margin  of  the  thorax  and  three  others  about  the  middle. 

The  colours  are  not  those  of  a  Stegomyia,  as  the  dark  is 
a  dark  brown,  and  the  white  is  not  the  silvery  white  of 
Stegomyia.  All  the  Mansonia  are  specially  liable  to  be 
infected  with  larval  ticks. 

Genera  of  Sub-family  JEdince  : — 

Theobald  now  makes  nine  genera  in  this  sub-family,  of 
which  only  the  more  important  are  here  given. 


,-EDIlNLE  231 

A.  Antennae  plumose  in  male.  Head  clothed 
with  narrow  curved  and  flat  scales.  Middle 
lobe  of  scutellum  with  six  border  bristles. 
Scutellum  with  narrow  curved   scales,  palpi 

in  ?  four-jointed,  in  $  two-jointed  JEdes. 

B.  Head  clothed  with  flat  scales  only. 

(a)  Fork  cells  normal  length. 

(1)  Mid-lobe  of  scutellum  with  four 
border  bristles :  palpi  of  5  two- 
jointed.     Small  dark  species   Verallina. 

Palpi  of  ?  five-jointed,  metallic    H<zmogogus. 

(2)  Mid-lobe  of  scutellum  with  six 
border  bristles  :  palpi  of  5  three- 
jointed  Skusea. 

{b)  Fork  cells  small. 

Scutellar   scales   narrow   curved,    wings 

with  Mansonia-like  scales  JEdeomyia. 

Genera  of  Sub-family  Limatiuce  : — 

Only  one  genus,  viz.,  Limatus,  occurs  in  this  sub-family. 
Head,  thorax,  and  scutellum  with  flat  scales. 

Metanotum  with    both  hairs  and  scales. 

Wings    with    broad,    elongated     conical 

scales.     Proboscis  in  $  elbowed  with  two 

seal y  tufts Limatus. 

Genera  of  Sub-family  Deinoceratince  : — 
Second  segment  of  antennas  very  long.  Palpi 
short  in  both  sexes.       Both    $    and    ? 
antennae  pilose. 

(1)  Eyes     well     separated  :     clypeus 

with  bristles    Deinocerites. 

(2)  Eyes  contiguous,  clypeus  without 
bristles,   second  joint  of  antennas 

very  long  indeed Di7iomimetes. 

Genera  of  Sub-family  Uranotcenince  : — 
A.  First  fork  cell  very  small. 
(1)  Clypeus  nude. 

(a)  Male  ungues  normal.  Wings  with 
broad  lanceolate  lateral  scales  :  no 

inflated  ones  Uranotcettia. 

Wings    with    some    inflated   vein 

scales   Pseudoiiranotania. 

{b)  Male  ungues  broad  and  plate-like     Anisocheleomyia. 


232  JEDINM 

(2)  Clypeus  with  long,  dense  scales    Squamomyia. 

B.  Fork  cells  moderate  size. 

(1)  Proboscis  normal. 

(a)  First  fork  cell  normal  :  nearly  as 

large  as  the  second  fork  cell    Mimomyia. 

(b)  P'irst  fork  cell  expanded  basally  ...     Pseudograbhamia. 

(2)  Proboscis  swollen  apically,  elbowed  with 

complex  arrangement  of  hairs,  clypeus 

elongate,  almost  covering  palpi    Harpagomyia. 

(3)  Proboscis  and  clypeus   normal :    lateral 

vein  scales  forked  apically Hodgesia. 

C.  Fork  cells  short  but  first  longer  than  second. 

Proboscis  normal,  like   Uranotcenia  :  no  flat 
thoracic  scales Ficalbia. 

Genera  of  Sub-family  Dendromyince  : — 

A.  Legs  with  paddle-like  structures    Sabethes. 

B.  Legs  without  paddle-like  structures. 

(1)  Lateral    vein   scales    linear.     Proboscis 

longer  than  body     Phonio7nyia. 

Proboscis  shorter  than  body,  swollen  at 

apex    Wyeomyia. 

Metanotum  with  white  scales    Menolepis. 

(2)  Long,  dense  lateral  vein  scales  on  fifth  vein  Bolbodeomyia. 

(3)  Lateral  vein  scales  obovate  or  spatulate. 

Proboscis  fine  at  apex,  same  length  as 
abdomen  :  posterior  and  mid-cross 
veins  in  one  line  SabetJioides. 

Proboscis  short,  apex  swollen  :  pos- 
terior cross  vein  slightly  nearer  base 
than  mid.  Scales  of  mesonotum 
very  brilliant.  Metathorax  with 
scales  :  proboscis  same  in   3  and  ?      Sabe/hinus. 

Scales  of  metanotum  dusky  metallic, 

clypeus  without  scales    Dendromyia. 

Clypeus  with  scales     Prosopo/epis. 

Metanotum  nude.  Resembling  Den- 
dromyia, but  head  with  narrow 
curved  scales  in  middle  and  scutel- 
lum  with  narrow  curved  scales Philodcndromyia. 

Head  scales  flat  except  a  row  of  narrow 
curved  ones  behind  :  scutellum  with 
fiat  scales.  Culex  venation  and 
scales:  apex  of  abdomen  bristly  ...     Polylepidowyia. 


EGGS — LARV.E  233 

These  sub-families  include  a  large  number  of  genera 
and  species  which  differ  very  markedly  from  each  other 
as  well  as  from  the  Culicince.  Few  of  them  frequent 
towns  or  houses,  and  many  of  them  are  bush,  jungle,  or 
swamp  mosquitoes.  Some  of  them  attack  man  in  their 
free  state,  and  many  bite  only  or  mainly  in  the  daytime. 
They  have  been  less  studied  than  Culicince  and  Anophe- 
lince,  and  are  not  known  to  carry  disease.  The  fre- 
quency with  which  malaria  is  acquired  in  some  jungle 
districts  where  Anophelines  are  not  abundant  suggests 
the  possibility  that  some  of  these  mosquitoes  may  act  as 
carriers  of  malaria.  Experimental  evidence  is  wanting 
and  is  difficult  to  obtain,  as  most  of  these  mosquitoes 
will  not  feed  in  captivity  or  when  bred  from  larvae,  and 
many  of  them  die  rapidly  in  captivity.  Some  species 
are  so  susceptible  to  a  dry  atmosphere  that  they  will  die 
in  a  few  hours  after  their  removal  from  the  jungle. 

In  nature  they  rarely  leave  the  jungle  in  which  they 
live.  This  is  very  marked  with  some  species,  so  much  so 
that  standing  at  the  edge  of  a  clearing,  one  hand  thrust 
into  the  jungle  will  be  covered  with  mosquitoes,  whilst 
the  other  hand  in  the  open  will  not  be  attacked  at  all. 

The  eggs  of  only  a  few  species  of  these  mosquitoes  are 
known.  Frequently  they  form  raft-like  masses,  differing 
from  those  of  Culex  fatigans  in  that  they  are  less  compact. 

The  known  larvae  all  have  a  respiratory  siphon,  which 
may  be  very  short  and  broad,  and  resemble  Stegomyia 
larvae,  or  may  be  very  long  and  thin.  In  some  instances 
the  respiratory  siphons  are  profusely  ornamented  with 
simple  or  compound  hairs.  The  abdominal  and  thoracic 
segments  may  be  similarly  adorned. 

The  larvae  of  some  genera  lie  nearly  horizontally  at  the 
surface  of  the  water  when  at  rest,  and  are  frequently 
mistaken  for  Anopheline  larvae  if  the  respiratory  siphon  is 
not  observed. 

Many  of  these  mosquitoes  have  very  special  breeding 
places,  such  as  natural  collections  of  water  in  various 
plants,   pitcher  plants,    &c,   the  water  collected    in    the 


234  DIFFICULTIES 

joints  of  growing  bamboos  which  have  been  perforated 
by  coleopterous  and  other  larvae,  or  in  the  axils  of  leaves. 
Others  will  breed  freely  in  swamps,  puddles,  streams,  01 
at  the  edges  of  rivers. 

The  synopsis  should  enable  the  reader  to  distinguish 
the  chief  genera  common  in  any  part  of  the  world,  but 
is  far  from  being  complete.  For  full  information  the 
reader  is  advised  to  consult  the  "  Monograph  of  the 
Culicidce,"  Theobald,  vols,  iii.,  iv.  and  v.  For  distinction 
of  species,  the  size,  colouring,  and  particularly  the  mark- 
ings on  the  legs,  thorax  and  wings,  and  slight  modifica- 
tions in  the  arrangement  of  the  cross-veins  of  the  wings, 
become  important.  With  the  Anophelince  the  markings 
on  the  posterior  pair  of  legs  are  in  some  instances  sufficient 
for  the  identification  of  species. 

The  reader  is  warned  not  to  be  alarmed  at  the  apparent 
magnitude  of  the  subject.  It  is  true  that  this  table 
does  not  give  all  the  genera,  but,  on  the  other  hand,  many 
of  the  genera  are  of  limited  distribution,  and  in  few 
places  will  there  be  more  than  some  thirty  common 
species  of  mosquitoes,  which  can  readily  be  subdivided 
into  their  respective  genera  ;  and  often  the  species  can 
be  easily  identified  by  reference  to  the  standard  books — 
if  not  they  should  be  forwarded  to  England  to  one  of 
the  schools  of  Tropical  Medicine  for  identification.  For 
the  distinction  of  species  the  amateur  will  often  be  much 
helped  by  examination  of  the  eggs  or  larvae,  as  these 
sometimes  show  more  obvious  differences  than  the  adults. 

For  examination  of  the  external  characters  of  mosqui- 
toes the  method  of  mounting  on  entomological  pins  is 
in  every  way  preferable.  They  may  be  mounted  in 
Canada  balsam,  but  this  method  has  some  disadvantages, 
as  the  scales  are  rendered  too  transparent,  and  the  non- 
scaled  veins,  particularly  the  cross-veins,  are  difficult  to 
make  out.  Theobald  mounts  wings  and  other  parts 
separately  in  Canada  balsam. 

To  mount  in  Canada  balsam  the  insect  should  be 
placed  on  its  back  with  the  legs  separated  and  the  wings 


MOUNTING  235 

spread  out.  A  small  drop  of  thick  Canada  balsam  is 
placed  on  a  slide.  The  slide  is  then  held  in  the  hand 
so  that  the  drop  of  Canada  balsam  is  on  the  under  sur- 
face, and  this  is  gently  pressed  against  the  thorax  of  the 
mosquito,  and  the  mosquito  adheres  to  it  on  lifting 
it.  The  slide  should  then  be  turned  over  so  that  the 
mosquito  rests  on  it.  The  wings  and  legs  should  be 
arranged  to  taste,  and  a  drop  more  of  fluid  Canada 
balsam  placed  on  the  mosquito  ;  as  this  flows  over  the 
mosquito  it  will  cause  the  head  appendages  to  spread 
out.  A  mosquito  so  arranged  will  keep  indefinitely. 
To  complete  the  process  a  glass  ring  should  be  cemented 
round  the  mosquito,  and  when  firmly  set  the  cell  so 
formed  should  be  filled  with  balsam  and  a  cover-glass 
placed  over  it. 

Mounted  in  glycerine  jelly  the  mosquito  retains  its 
natural  colouring.  The  best  method  of  mounting  in 
glycerine  jelly  is  to  make  a  deep  cell  with  a  glass  ring 
and  slide.  Lay  the  mosquito  at  the  bottom  and  fill  the 
cell  with  the  jelly  previously  melted  by  placing  in  hot 
water.  Place  a  cover-glass  over  the  jelly,  taking  care  to 
avoid  air  bubbles.  It  is  very  difficult  to  arrange  a 
mosquito  in  the  jelly.  Specimens  mounted  in  this 
manner  often  have  their  legs  interlaced.  It  is  a  useful 
method  for  mosquitoes  which  have  been  kept  in  spirit, 
as  such  mosquitoes  are  too  brittle  to  stand  any  handling 
and  cannot  be  pinned  out. 

Specimens  so  mounted  should  be  ringed  with  some 
varnish  as  soon  as  the  jelly  is  set,  otherwise  evaporation 
will  take  place  and  the  jelly  shrink,  resulting  in  the 
formation  of  air  bubbles.  If  this  accident  should  happen 
the  cover-glass  must  be  removed  from  the  jelly  and  fresh 
glycerine  jelly  added.  This  is  easily  done  by  dissolving 
off  the  cement  and  gently  warming  to  melt  the  jelly. 
The  cover-glass  can  then  be  removed  and  fresh  jelly 
melted  and  added.  The  cover-glass  is  to  be  replaced, 
and  when  the  jelly  has  set  cemented  on. 

Another  method  of  mounting  mosquitoes,  known  as  the 
Bentley-Taylor  method,  is  as  follows  : — 


236  MOUNTING 

Prepare  (1)  a  solution  of  1  per  cent,  celloidin  in  absolute 
alcohol  ;  and  (2)  a  solution  of  celloidin  in  absolute 
alcohol  of  the  consistence  known  as  "  thick  "  in  ordinary 
histological  work. 

Catch  the  mosquito,  chloroform  it,  and  when  dead  or 
narcotised,  place  a  drop  of  solution  1 — thin  celloidin — 
on  a  cover-glass.  Place  the  insect  back  downwards  on 
the  cover-glass.  In  the  majority  of  cases  wings  and  legs 
spread  themselves  out  in  the  orthodox  exhibition  position, 
but  if  not  they  may  be  adjusted  with  a  needle. 

When  the  thin  solution  has  become  "tacky,"  i.e.,  in 
about  eight  or  ten  minutes,  place  a  drop  of  the  thick 
solution,  No.  2,  over  the  insect.  Invert  the  cover-glass 
over  a  hollow  slide  to  which  it  may  be  fixed  by  a  ring 
of  balsam.  The  specimen  is  then  complete  and  in  that 
condition  both  ventral  and  dorsal  surfaces  can  be 
examined. 


237 


CHAPTER    XII. 
Dissection    of    Mosquitoes. 

The  internal  anatomy  of  the  mosquito  is  not  very 
complicated,  and  the  more  important  parts  are  easily 
dissected  out. 

The  alimentary  canal  is  a  tube  with  dilatations  running 
from  the  proboscis  to  the  anus,  which  is  terminal.  The 
proboscis  is  suctorial  and  piercing  and  composed  of  the 


Fig.  104. — a,  Antennae  ;  cl,  clypeus ;  Ixe,  labrum-epipharynx  ;  mn,  man- 
dibles ;  hp,  hypopharynx  ;  mx,  maxillae  ;  la,  labium ;  mp,  maxillary  palps ; 
lab,  labella.     (After  Nuttall.) 

following  parts  (fig.  104)  :  (1)  Below  is  a  deeply-grooved, 
fleshy  labium  ;  this  contains  air  tubes,  is  covered  with 
scales,  and  forms  what  appears  to  be  the  proboscis,  as 
when  the  mosquito  is  at  rest  the  other  elements  are  con- 
tained in  the  groove.  It  terminates  in  two  small  jointed 
lobes — the  labellce.  (2)  Above  is  the  labrum,  or  more 
correctly,  the  labrum-epipharynx,  as  this  part  results  from 
the  fusion  of  the  labrum  and  epipharynx.     The  labrum 


=38 


MOUTH    PARTS 


is  deeply  grooved  on  the  under  surface  and  terminates 
in  a  sharp  point.  (3)  This  groove  is,  by  the  apposition 
of  the  hypopharynx,  converted  into  a  tube,  up  which  the 
food  is  sucked;  this  tube  is  continuous  with  the  cavity 
of  the  pumping  organ.  The  hypopharynx  is  a  flattened 
chitinous  rod  terminating  in  a  sharp  point  ;  it  is 
strengthened  in  the  middle  by  a  ridge,  and  in  this 
thickening  is  contained  a   minute  [tube,  the  termination 


Fig.  105. — a,  Antennse  ;  cl,  clypeus  ;  mp,  maxillary  palps  ;  Ixe,  labrum- 
epipharynx  ;  mn,  mandibles  ;  mx,  maxillae. 

of  the  salivary  duct.  Down  this  minute  tube  the  saliva 
is  ejected  into  the  depths  of  the  wound  made  by  the 
penetrating  parts  of  the  proboscis.  (4)  There  are  two 
pairs  of  piercing  organs,  the  mandibles  and  maxilla: ; 
both  of  these  are  thin  strips  of  chitin  with  sharp  cutting 
edges  terminating  in  a  lancet-like  point.  The  cutting 
edge  may  or  may  not  be  serrated.  In  most  species  the 
maxillae  only  have  a  serrated  edge  (fig.  105). 

In  the  male  the  mandibles  are  well  developed,  but  less 
so  than  in  the  female  ;  the  maxillae  are  absent  in  some 
species  ;  the  hypopharynx  is  closely  adherent  to  the 
labium  in  its  whole  length. 


MOUTH    PARTS  239 

Of  these  elements  the  labium  mainly  acts  as  a  sheath 
and  protects  the  more  delicate  parts  of  the  proboscis 
from  injury.  It  does  not  penetrate  the  skin.  The  tip 
is  applied  firmly  to  the  skin,  and  in  the  angle  between 
the  two  labellae  all  the  other  elements  of  the  proboscis 
are  thrust  into  the  skin  (fig.  106).  No  doubt  it  aids  in" 
penetration  by  keeping  together  and  rendering  more  rigid 
the  other  elements,  and  as  it  is  supplied  by  nerves  aids 
in  the  selection  of  a  suitable  place  for  puncture. 


Fn;.  106. — /?',  Labium  ;    la,  labella  ;    Ixe,  labrum-epipharynx  ;    mx,  maxillae  : 
mn,  mandibles. 


As  the  other  elements  penetrate  the  skin  the  labium 
becomes  bent  on  itself,  as  depicted  in  the  diagram. 

The  penetrating  elements  form  two  tubes,  with  the 
mandibles  and  maxillae  at  the  sides.  Up  the  superior 
tube  formed  by  the  groove  of  the  labrum  epipharynx 
and  the  flat  hypopharynx  the  blood  is  sucked,  whilst  the 
saliva  is  ejected  through  the  small  tube  in  the  hypo- 
pharynx  (fig.  107). 

The  main  points  in  the  anatomy  of  the  proboscis  can 
be  readily  demonstrated.  The  hypopharynx  often  closely 
adheres  to  the  labrum-epipharynx,  so  that  it  is  the  most 
difficult  component  to  separate  and  identify. 


>40 


DISSECTION 


To  demonstrate  the  two  tubes  formed  by  the  apposition 
of  these  elements,  transverse  sections  of  the  proboscis  are 
requisite.  From  the  tube  thus  formed  by  the  labrum- 
epipharynx  and  hypopharynx  the  blood  is  conveyed  into 
the  pumping  organ,  which  is  composed  of  three  chitinous 
plates,  to  which  muscles  are  attached.  This  in  turn 
forces  the  blood  into  a  membranous  tube  which  is  con- 
tinuous with  the  commencement  of  the  oesophagus. 
These  parts  also  can  only  be  satisfactorily  demonstrated 
in  sections. 


4-  mx. 


sa/ 


Fig.  107. — !xe,  Labrum-epiphaiynx  ;  mn,  mandibles  ;  hp,  hypopharynx  ;  sal, 
salivary  duct ;  ir,  tiachea  ;  tines,  muscle  ;  »ix,  maxillae. 


The  rest  of  the  alimentary  canal  is  best  shown  by 
dissections. 

Dissection. — Only  freshly  killed  mosquitoes  are  suitable 
for  dissection.  They  can  be  killed  in  many  ways. 
With  those  required  for  dissection  no  great  precaution 
need  be  taken,  as  it  is  immaterial  if  the  scales  are  knocked 
off.  They  can  be  killed  with  tobacco  (cigarette)  smoke, 
chloroform  vapour,  or  stunned  by  concussion. 

Dissection  of  Alimentary  Canal. — The  mosquito  should 
be  caught  in  a  test  tube.  This  is  done  by  placing  the 
test  tube  slowly  over  a  resting  mosquito.  If  it  is  done 
rapidly  the  mosquito  will  take  alarm  and  usually  escape. 


DISSECTION  241 

It  is  important  to  so  approach  the  mosquito  that  no 
shadow  falls  on  it.  By  proceeding  cautiously  mosquitoes 
are  readily  caught  in  this  way,  and  they  then  fly  to  the 
closed  end  of  the  tube.  With  practice  nine  or  ten 
mosquitoes  can  be  caught  in  this  manner  in  one  tube. 

Six  clean  slides  and  cover-glasses  should  be  prepared, 
and  on  three  of  them  a  drop  of  normal  saline  solution 
should  be  placed ;  the  other  three  should  be  left  dry. 
Two  sharp  needles  are  also  required. 

After  killing  or  stunning  the  mosquito  it  should  be 
transfixed  through  the  thorax  with  a  mounted  needle  and 
the  legs  and  wings  pulled  off  and  dropped  on  a  clean  dry 
slide.  This  can  be  easily  done  with  the  fingers,  but  there 
is  no  objection  to  the  use  of  forceps.  These  can  be 
examined  dry  by  covering  with  a  cover-glass  and  fixing 
this  with  gummed  paper,  or  they  can  be  mounted  in 
glycerine  jelly  or  Canada  balsam. 

The  mosquito,  denuded  of  its  limbs,  is  placed  in  the 
saline  solution  on  one  of  the  slides.  The  posterior  part 
of  the  abdomen  is  gently  flattened  with  the  shaft  of  a 
needle  and  two  nicks  made,  one  on  each  side,  about  the 
junction  of  the  second  and  third  last  segments  (fig.  108). 
This  weakens  the  exoskeleton  at  that  point  so  much  that 
when  traction  is  made  on  the  last  segment  the  exoskeleton 
breaks. 

Traction  is  best  exercised  by  fixing  with  the  point  of 
one  needle  the  thorax  and  laying  the  other  flat  on  the 
last  segment  and  steadily  and  slowly  dragging  away  from 
the  head. 

In  the  space  between  the  broken  ends  of  the  exoskele- 
ton a  series  of  white  strands  will  be  seen — the  intestine 
and  Malpighian  tubes.  On  further  traction  the  stomach 
and  part  of  the  oesophagus  will  appear  (fig.  109). 

If  the  traction  be  continued  from  the  end  there  is  a  risk 
that  the  stomach  may  break  off.  It  is  better  to  shift 
the  needle  from  the  posterior  segments  to  the  oesophagus 
at  the  point  of  emergence  from  the  broken  end  of  the 
abdomen,  and  pull  slightly  obliquely  on  this  so  as  to  drag 
16 


242 


DISSECTION 


Fig.  108. 


DISSECTION 


243 


the  rest  of  the  oesophagus  out  of  the  abdomen  and  thorax. 
It  should  be  covered  with  a  cover-glass. 

The  stomach  with  its  appendages  can  now  be  examined 
directly.  The  genital  organs  will  be  still  attached  to 
the  terminal  segments  of  the  mosquito  and  can  be 
examined  at  the  same  time.  To  show  them  completely 
it  is  better  under  the  microscope  to  tease  off  the  remainder 
of  the  exoskeleton  of  the  last  two  segments. 


The  remainder  of  the  mosquito  should  be  placed  in 
the  drop  of  saline  solution  on  one  of  the  other  slides 
for  the  dissection  of  the  salivary  glands. 

To  dissect  the  salivary  glands  there  are  several  methods. 
The  one  described  here  is  that  which  is  most  readily 
learnt   and    by  which   uniform   results    can    be   obtained 


244  SALIVARY   GLANDS 

fairly  readily.  It  has  the  disadvantage  that  other  tissues 
are  present  in  the  dissection  and  may  conceal  more  or 
less  of  the  lobes. 

The  principle  is  to  take  as  small  a  portion  of  the 
mosquito  as  is  possible,  with  the  certainty  that  the 
portion  contains  both  salivary  glands. 

The  chitinous  portion  of  this  remnant,  which  includes 
the  bases  of  two  pairs  of  legs,  is  sufficient  to  conceal  the 
glands  ;  it  must  therefore  be  broken  up  with  the  points 
of  the  needles  into  four  or  five  fragments,  which  should 
be  about  a  quarter  of  an  inch  from  each  other.  These 
fragments  are  of  course  still  in  the  saline  solution. 

A  cover-glass  should  be  placed  over  the  whole  series 
of  fragments  and  each  portion  should  be  compressed  in 
turn  with  the  point  of  the  needle.  In  the  great  majority 
of  instances  the  salivary  glands  will  be  squeezed  out  from 
under  the  portions  of  the  exoskeleton.  It  is  common  to 
find  a  small  portion  of  one  lobe  still  covered  by  the 
exoskeleton. 

Another  method  is  to  squeeze  the  contents  of  the 
thorax  out  towards  the  head  end  of  the  thorax,  after 
cutting  off  the  head,  when  the  salivary  glands  may  be 
shot  out  uninjured.  This  method  is  uncertain,  and  some 
of  the  lobes  are  often  damaged,  but  when  successful  the 
glands  are  sometimes  better  displayed  and  have  less 
surrounding  tissues  than  the  other  method  (fig.  no). 

The  first  method  recommended  here  has  the  very 
decided  disadvantage  that  the  salivary  glands  are  not 
isolated  but  surrounded  by  other  tissues.  For  per- 
manent preparations  it  is  not  a  good  method,  as  other 
tissues  are  present.  The  glands  do  not  dry  quickly  and 
become  fixed  to  the  slide  as  isolated  glands  do. 

For  mere  examination  it  is  satisfactory,  but  when 
confidence  has  been  acquired  by  this  method,  if  per- 
manent preparations  are  desired  the  salivary  glands  must 
be  isolated. 

In  the  best  method  for  isolation  of  the  glands  the 
head  is  not  cut  off,  but  the  back  of  the  thorax  is  separated 


SALIVARY   GLANDS  245 

by  ;i  longitudinal  incision.  A  sharp  edge,  such  as  is 
provided  by  a  surgical  needle  or  cataract  knife,  is  better 
than  an  ordinary  needle.  A  second  incision  at  right 
angles  to  the  first  is  made  at  the  level  of  the  second 
pair  of  legs.  The  head  is  now  transfixed  as  near  the 
neck  as  possible  with  one  needle  and  the  remnant  of  the 
thorax  fixed  with  another.  On  pulling  on  the  head 
the  salivary  glands  will  be  pulled  out  of  their  bed  in  the 
thorax  and  can  be  seen  attached  to  the  head.  Micro- 
scopic examination  under  a  low  power  objective  is 
necessary  at  this  stage.  A  final  cut  will  separate  the 
head,  and  the  salivary  glands  are  left  isolated  (fig.  111). 
It  is  not  uncommon  to  find  that  the  ends  of  some  of  the 
lobes  have  been  left  behind  in  the  thorax  or  the  glands 
otherwise  damaged,  but  perfect  specimens  can  be  obtained 
in  this  way. 


Fig.  1 10. 

The  excess  of  salt  solution  should  be  removed  with 
blotting-paper,  the  specimen  air-dried,  fixed  in  alcohol, 
and  stained  on  the  slide. 

To  show  the  relations  of  these  parts  and  other  structures 
in  the  mosquito  serial  sections  are  requisite.  The  mos- 
quito can  be  cut,  embedded  either  in  celloidin  or  in 
paraffin  wax. 

To  show  structure,  young  mosquitoes  which  have  only 
been  hatched  for  a  few  hours  are  best,  and  they  should 
be  placed  alive  in  spirit  and  hardened  in  absolute  alcohol. 

With  older  mosquitoes  it  is  better  to  puncture  the 
thorax  and  abdomen  with  the  point  of  a  fine  sharp  knife 
or  needle,  so  as  to  facilitate  the  entrance  of  the  paraffin 
or  celloidin. 


246 


SECTIONS   OF   MOSQUITOES 


To  Cut  and  Stain  Sections  ok  Mosquitoes. — 
According  to  Dr.  Low  the  best  method  is  to  kill  the 
mosquitoes  by  dropping  them  into  60  per  cent,  alcohol 
alive,  so  that  some  spirit  may  be  drawn  into  the  interior. 
Keep  them  live  days  in  this  spirit.  Remove  the  wings, 
and  legs  from  the  mosquito  and  place  the  trunk  in  95 
per  cent,  alcohol  for  twenty-four  hours,  then  in  absolute 
alcohol  for  twenty-four  hours,  then  in  alcohol  and  ether 
equal  parts  twenty-four  hours.  After  this  thin  celloidin 
one   day,   thick   celloidin    one    day.      Mount    on   blocks, 


Fig.  hi. 


hardening  the  celloidin  on  them  in  60  per  cent,  spirit; 
then  cut  serial  sections,  keeping  the  sections  in  60  per 
cent,  spirit.  For  staining  float  out  in  water.  Stain  in 
watch  glasses  with  haemalum  or  haematoxylin  for  live  to 
ten  minutes  so  as  so  overstain  decidedlv.  Decolorise 
with  1  per  cent,  hydrochloric  acid  in  70  per  cent,  alcohol 
till,  when  replaced  in  water,  only  a  faint  violet  colour  is 
retained  by  the  mosquito.  Replace  in  60  per  cent,  spirit, 
then  in  95  per  cent.,  and  from  that  to  carbol-xvlol — 25  per 


SECTIONS   OF   MOSQUITOES  247 

cent.,  till  the  section  appears  perfectly  clear  and  trans- 
parent. Transfer  to  slide,  press  firmly  with  clean  filter 
paper,  and  mount  in  xylol  balsam. 

For  mosquitoes  celloidin  is  to  be  preferred,  particularly 
for  the  demonstration  of  filaria  in  situ.  For  structure, 
good  results  can  also  be  obtained  with  paraffin  sections. 
For  this  purpose  recently  hatched  mosquitoes  are  the 
best,  and  they  should  be  placed  alive  in  the  spirit,  passed 
through  the  usual  processes,  embedded  in  paraffin  and 
serial  sections  cut.  These  small  sections  are  easily  de- 
tached from  the  slide.  The  method  recommended  by 
Annett  and  Dutton  to  prevent  this  is  to  lay  the  paraffin 
section  on  a  thin  layer  of  two  parts  of  liquid  glucose  and 
one  part  of  a  thick  syrup  of  pure  dextrin,  spread  on  a  slide 
and  kept  in  the  hot  incubator  till  the  glucose  mixture  has 
dried  hard.  The  paraffin  is  then  removed  by  xylol  and 
alcohol,  and  a  solution  of  photoxylin  is  poured  over  the 
slide  so  as  to  form  a  film  over  the  sections.  This  is 
allowed  to  set  till  the  edges  of  the  photoxylin  film  crinkle. 
On  placing  the  slide  in  water  the  film  comes  away  with 
the  sections,  which  can  then  be  stained  in  the  usual  way 
as  recommended  for  celloidin  sections.  Carbol-xylol 
must  be  used  for  clearing. 

In  dissections  the  points  to  be  observed  are  as  follows  : — 

At  the  commencement  of  the  oesophagus  are  three 
diverticula,  of  which  one,  the  ventral,  is  much  larger  than 
the  others.  These  diverticula  usually  contain  air  and 
sometimes  food.  They  vary  greatly  in  size.  Thev  are 
often  pulled  out  of  the  thorax  with  the  oesophagus,  but  to 
show  them  satisfactorily  it  is  necessary  to  tear  off  the  back 
of  the  thorax  and  break  through  the  upper  segments  of 
the  abdomen  before  exercising  traction  on  the  oesophagus. 
Bacteria  in  large  numbers  are  found  in  these  diverticula. 

The  stomach  is  seen  as  a  clear  translucent  expansion  of 
the  oesophagus.  The  cells  lining  the  intestine  appear  to 
be  polygonal,  and  the  outlines  can  be  clearly  made  out  by 
using  central  light  only. 

At  the  hinder  end  of  the  stomach  just  before  the  junction 


248  ANATOMY   OK   THE    MOSQUITO 

buccal  cavity 
pharynx  c_ 

dorsal  reservoirs 


oesoph.  valve 
and  caeca 


midgut 
begins 


stomach  - 
malphigian  tube 

midgut  ends  -  - 
ileum   -  - 

colon 

rectum 


Fig.   112. — Internal  Anatomy  of  the  Mosquito. 


APPEARANCES  249 

with  the  hind  gut,  are  seen  the  five  Malpighian  tubes, 
which  are  much  more  opaque  and  are  lined  by  large 
nucleated  cells,  and  these  cells  contain  granules  or  droplets 
of  a  retractile  oily  nature.  The  lumen  of  these  diverticula 
is  difficult  to  make  out  (tig.  112). 

The  continuation  of  the  intestine  is  a  tube  which  is 
not  straight — the  hind  gut  or  rectum.  The  cells  vary  in 
different  parts  of  the  tube  and  the  variation  differs  in 
different  species.  Parasites  of  various  kinds  may  be 
found.  Examination  with  a  high  power,  one-twelfth 
inch,  is  necessary,  as  the  youngest  forms  of  the  malarial 
parasite  cannot  be  readily  seen  with  lower  powers,  and 
therefore  familiarity  with  the  normal  appearance  of  the 
cells  of  the  mosquito's  stomach  with  this  power  is 
essential. 

Some  confusion  may  be  caused  by  the  air  tubes  which 
ramify  over  the  surface  of  the  stomach.  These  appear  to 
be  black  when  seen  by  transmitted  light,  on  account  of 
the  air  they  contain,  but  silvery  white  when  seen  by  re- 
flected light  for  the  same  reason.  They  can  be  recognized 
by  the  spiral  thickening  and  their  repeated  branching. 

The  cells  seen  in  the  stomach  form  the  epithelial  lining 
of  that  organ.  They  are  detached  by  pressure  on  the 
stomach.  By  making  a  nick  in  the  side  of  the  stomach  and 
alternately  floating  up  the  cover-glass  with  water  and  ab- 
stracting it  on  the  other  side  of  the  cover-glass  with  blotting 
paper,  the  detached  epithelium  can  be  removed.  By  re- 
peating this  process  several  times  the  epithelial  lining  can 
not  only  be  detached  but  in  great  part  washed  away.  This 
measure  may  be  required  to  wash  out  the  contents  of  the 
stomach,  particularly  when  they  are  dark  and  opaque  with 
altered  blood.  It  is  also  necessary  for  satisfactory  staining 
of  malaria  parasites  in  the  wall  of  the  stomach. 

When  the  epithelium  is  washed  out  the  stomach  is 
reduced  to  a  clear  transparent  bag.  Longitudinal  and 
transverse  markings  are  often  seen  in  this,  and  are  indi- 
cations of  the  muscular  bands. 

In   a   stomach    with   the  epithelium   thus  removed    the 


250  SALIVARY   GLANDS 

developed  malaria   parasites  can    be  stained  by   running 
the  stains  under  the  cover-glass.     Picrocarmine  gives  fair 

results.      When  sufficiently  stained  the  excess  of  stain  can 

be  washed  out  in  the  same  manner,  and  finally  Farrant's 

solution  run  in  to  displace  the  water. 

The  stomach  with  the  epithelium  intact  can  also  be 
stained  in  this  maimer,  but  more  uniform  staining  is 
obtained  by  removing  the  cover-glass  and  allowing  the 
stomach  to  dry  on  the  slide.  It  can  then  be  fixed  in 
alcohol  and  stained  with  any  basic  stain,  and  after 
washing,  dehydrated  in  alcohol,  cleared  with  xylol,  and 
mounted  in  Canada  balsam.  By  this  method  the  de- 
veloped malarial  parasites  are  not  well  shown,  as  they  will 
not  stand  drying  or  dehydration  without  great  distortion. 

The  salivary  glands  can  be  mounted  in  the  same  way, 
but  in  the  Farrant's  solution  the  cells  wrinkle  and  poor 
results  are  obtained.  Somewhat  better  results  are  ob- 
tained by  removing  extraneous  tissues  under  the  micro- 
scope and  drying  the  slide  in  the  air.  The  salivary 
glands  can  then  be  fixed,  stained  and  mounted.  In  the 
fresh  preparation  the  cells  will  be  found  to  vary  greatly, 
and  they  are  often  distended  with  retractile  droplets. 
These  may  be  so  numerous  as  to  fill  many  or  all  of  the 
cells.  The  cells  in  the  middle  lobe  are  smaller  and  often 
differ  in  appearance  from  those  in  the  lateral  lobes. 
The  main  duct  has  cubical  epithelium,  which  is  continued 
for  some  distance  down  the  lobules.  In  Anopheles  the 
ends  of  the  ducts  in  the  lobules  are  dilated,  whilst  in  most 
of  the  genera  the  ducts  maintain  the  same  calibre  in 
their  entire  length.  Occasionally  a  diverticulum  is  met 
with.  This  may  be  terminal,  so  that  the  lobule  bifur- 
cates at  the  end,  or  it  may  be  found  in  any  other  part. 
In  Psorophora  each  gland  has  five  lobes. 

At  first  there  may  be  difficulty  in  finding  these  glands 
with  a  low  power.  The  point  to  search  for  is  die  main 
duct  and  its  trifurcation,  as  this  is  most  readily  seen  even 
if  the  gland  is  embedded  in  muscular  or  other  tissues. 
To  see  the  character  of  the  glandular  cells  in  detail  a  3V, 


GENITAL   ORGANS  25  I 

oil  immersion  must  be  used,  and  the  diaphragm  nearly 
closed,  as  the  cells  are  very  transparent. 

Sporozoa  have  been  described  in  the  ovaries  of  the 
mosquito,  and  we  know  that  the  piroplasma  of  cattle  and 
dogs  are  transmitted  by  an  infected  tick  to  its  offspring. 

In  the  present  state  of  our  knowledge  it  is  therefore 
advisable  to  study  the  internal  genital  organs  of  the 
mosquito  to  some  extent.  These  are  usually  removed 
with  the  stomach,  but  in  part  are  hidden  by  the  exo- 
skeleton  of  the  last  two  or  three  segments  of  the  mosquito 
which  remain  still  attached  to  the  stomach.  This  exo- 
skeleton  can  be  teased  off  with  a  pair  of  needles  ;  this  can 
be  done  under  a  dissecting  or  other  microscope  with 
greater  certainty  of  not  at  the  same  time  injuring  the 
genital  organs. 

The  female  genital  organs  consist  of  a  pair  of  ovaries 
opening  into  a  common  tube  by  the  ovarian  tubes.  Into 
this  common  tube  opens  a  mucous  gland  and  also  the 
spermathecce  by  a  long  narrow  duct.  The  spermathecas 
are  chitinous  sacs  and  store  up  the  spermatozoa  received 
from  the  male.  In  this  way  by  a  single  act  of  coitus  by 
the  male  sufficient  spermatozoa  are  stored  up  to  enable 
many  series  of  eggs  to  be  fertilized.  The  number  of 
spermathecas  varies.  In  most  genera  there  are  three,  but 
in  the  Anophelince  there  is  only  one  and  in  Mansonia  two. 

The  male  genital  organs  consist  of  two  testicles  joined 
by  vasa  deferentia  to  the  ejaculatory  duct  formed  by  their 
union.  Just  before  this  junction  each  vas  deferens  is 
connected  by  a  short  tube  with  a  sac-like  receptacle — the 
vesicula  seminalis. 

The  ejaculatory  duct  leads  to  a  short  fleshy  penis 
situated  between  two  internal  claspers,  internal  gonapo- 
physes,  and  on  each  side  of  these  are  the  large  conspicuous 
external  claspers. 

The  spermatozoa  are  rounded  bodies  with  a  flagellum. 
According  to  Giles  they  do  not  reach  their  full  develop- 
ment in  the  male,  but  in  the  spermathecae  of  the  female. 


2  c  2 


CHAPTER    XIII. 

Demonstration   of    Development   of    Parasites    in 
Mosquitoes. 

In  the  freshly  shed  blood  we  saw  that  so-called  sexual 
forms  of  the  parasites  of  malaria — gametocytes — occurred 
which  flagellated,  and  that  forms  differing  little  from 
these  did  not.  However  much  the  blood  was  altered  by- 
exposure  to  air,  water,  or  in  the  mosquito's  stomach,  a 
proportion  of  non-flagellating  bodies  was  always  present. 
Both  those  that  flagellate  and  those  that  do  not  are  the 
gamete  or  sexual  forms  of  the  parasite.  They  are  only 
easily  recognized  in  autumno-aestival  fever  (sub-tertian), 
where  they  appear  as  the  crescent  bodies.  It  is  simpler 
to  follow  the  development  in  that  species  of  parasite  on 
this  account,  though  the  same  changes  occur  in  the  other 
species  of  malaria. 

In  the  shed  blood  the  crescents  rapidly  undergo  changes 
if  the  blood  be  exposed  to  the  air  or  moisture  be  added 
to  it.  If,  on  the  other  hand,  air  and  moisture  be  excluded 
no  change  occurs  in  the  crescents  till  they  die  and  break- 
up. To  exclude  the  air  a  drop  of  vaseline  is  placed  on 
the  finger-tip,  and  the  ringer  is  pricked  through  it  so  that 
a  drop  of  blood  exudes  into  the  centre  of  the  oil.  The 
oil  and  the  contained  drop  of  blood  are  transferred  to 
a  slide  and  the  whole  compressed  under  a  cover-glass. 
The  blood  can  be  watched  indefinitely  and  no  change 
will  be  found  to  occur  in  the  crescents  till  they 
disintegrate. 

If,  however,  a  drop  of  blood  is  taken  up  on  a  cover- 
glass  and  exposed  freely  to  air  for  two  minutes  and  then 


CONJUGATION — ZYGOTES  253 

placed  on  a  slide,  flagellating  forms  will  rapidly  appear 
and  the  crescents  which  do  not  flagellate — the  females — 
will  become  round. 

Instead  of  freely  exposing  to  air,  admixture  with  water 
leads  to  the  same  result.  This  can  be  conveniently  done 
by  breathing  on  the  slide  before  placing  the  cover  and 
drop  of  blood  on  it. 

In  short,  a  change  in  the  environment  of  the  sexual 
forms  of  the  parasites  which  does  not  kill  them  leads  to 
transformations  due  to  their  becoming  sexually  active. 
The  same  changes  take  place  in  the  stomach  of  the 
mosquito  with  greater  certainty  and  rapidity. 

To  demonstrate  satisfactorily  these  changes  it  is  neces- 
sary to  have  a  fairly  good  crescent  infection.  As  has 
been  already  seen,  the  flagella  from  the  males  are  actively 
motile,  and  these  are  the  sexually  active  agents  which 
enter  the  female  and  fertilize  it.  The  process  is  one  of 
conjugation,  and  the  product  is  a  zygote.  At  first  it  is 
an  actively  motile  body,  termed  the  travelling  vermicule 
or  ookinct.  This  travelling  vermicule  contains  the  pig- 
ment of  the  female  crescent  and  is  pointed  at  one  end. 

In  the  stomach  of  a  suitable  mosquito — several  species 
of  Anophelina  for  human  parasites  and  Culex  fatigans  for 
proteosoma — the  vermicule  passes  out  of  the  stomach 
cavity  and  the  zygote  becomes  encysted  in  the  stomach 
wall.  About  thirty-six  hours  after  feeding  on  an  infected 
person  these  encysted  zygotes  will  be  found,  and  can  be 
readily  recognized  by  their  pigment,  which  at  this  stage 
can  be  seen  to  be  little  changed  from  the  pigment  of 
the  parasites  from  which  they  were  derived.  They  are 
best  seen  in  fresh  specimens,  but  can  be  stained  with 
any  basic  stain  and  seen  after  the  epithelium  has  been 
removed  from  the  stomach.  The  youngest  forms  are 
a  little  larger  than  a  red  blood  corpuscle,  but  they 
rapidly  increase  in  size,  though  at  a  rate  varying  with 
the  temperature  of  the  air.  At  about  8o°  F.  they  attain 
their  full  development  in  Myzomyia  funesta  in  twelve 
days.     In   some  species  of   AiioplicJina,  under  the  most 


254  ZYGOTES — SPOROZOITES 

favourable    conditions,    the    full    development    may    take 
place  in  eight  days. 

With  this  increase  in  size  there  is,  of  course,  no  increase 
in  the  pigment,  as  the  zygote  does  not  derive  its  nutriment 
from  the  blood.  The  pigment  therefore  is  relatively 
scanty  and  absorption  or  solution  of  it  must  take  place, 
as  it  frequently  disappears  completely. 

When  fully  grown  the  zygotes  attain  the  size  of  50  or 
60  /i.  The  growth  of  the  parasites  is  entirely  outwards 
into  the  body  cavity  of  the  mosquito  and  away  from 
the  lumen  of  the  intestinal  tube,  so  that  when  mature 
they  appear  to  be  globular  excrescences  stuck  on  to  the 
stomach.  The  proportion  of  gametocytes  that  form 
zygotes  varies  a  great  deal.  When  several  mosquitoes 
have  fed  at  the  same  time  and  all  apparently  fed  well,  in 
some  there  will  be  no  zygotes,  in  others  two  or  three, 
whilst  some  may  have  fifty  or  more.  Darling  used  blood 
in  a  case  in  which  twenty-two  crescents  were  present  to 
100  leucocytes.  He  found  that  the  average  increase  of 
the  mosquitoes  after  feeding  was  about  *ooi  grm.  and 
as  the  leucocytes  were  6,500  per  c.mm.,  which  gives 
some  1,088  gametes  ingested,  or  as  a  result  of  three 
feedings  if  half  were  males  and  half  females  there  should 
have  been  1,632  zygotes.  There  were  only  50,  indicating 
a  loss  of  97  per  cent.  The  loss  appears  to  be  mainly 
due  to  phagocytosis  by  polymorphonuclear  leucocytes. 

The  contents  of  the  zygote  first  divide  into  a  series 
of  segments  called  sporoblasts  or  blastophores.  These 
blastophores  soon  lose  their  smooth  outline  and  have  an 
irregular  shaggy  appearance,  which  as  they  become  more 
mature  is  seen  to  be  due  to  the  conversion  of  the  outer 
part  of  the  blastophore  into  a  mass  of  filaments  attached 
by  one  end  to  a  small  central  residual  mass.  When  quite 
mature  these  filaments  break  off  and  the  cyst  is  then  filled 
with  these  filaments,  which  are  narrow  bodies  pointed  at 
both  ends  and  about  14  \x  in  length.  These  bodies  are 
known  as  sporozoitcs.  Zygotoblasts,  blasts,  exotospores, 
are  names  that  have  also  been  employed. 


SPOROZOITES — SALIVARY    GLANDS 


255 


In  the  fresh  state  they  can  be  seen  only  in  specimens 
immersed  in  saline  solution  or  in  weak  1  per  cent, 
formalin  solution. 

To  observe  them  the  freshly  dissected  stomach  in  one 
of  these  solutions  is  covered  with  a  cover-glass,  and  by 
gently  moving  this  cover-glass  with  a  needle  the  stomach 
can  usually  be  rolled  over  a  little  so  that  one  of  the 
mature  zygotes  is  seen  in  profile  projecting  from  the  edge 
of  the  stomach  (fig.  113).  Pressure  with  a  needle  on  the 
cover-glass  will  now  cause  the  rupture  of  the  capsule  of 
the  zygotes  and   the  contents,  the  blasts   or  sporozoites 


Fig.     113. 


will  be  poured  into  the  surrounding  saline  solution  and 
can  then  be  examined.  If  quite  mature  the  contents 
will  be  entirely  composed  of  sporozoites  with  a  few  small 
round  masses  of  residual  protoplasm,  and  in  some  cases 
a  few  small  grains  of  pigment  that  have  escaped  absorp- 
tion. 

If  not  quite  mature  some  of  the  sporozoites  will  remain- 
attached  to  the  protoplasmic  residue  which  formed  the 
centre  of  the  blastophore,  forming  a  tangled  mass  round 
this  centre. 

If   empty    cysts   are    found    attached    to   the   stomach 


256  DEVELOPMENT  OF    FILARIA 

detached  sporozoites  will  be  found  in  the  fluids  from 
any  part   of  the  body  of  the  mosquito,  and  in  some  ot 

the  cells  in  the  salivary  glands  they  will  be  found  in 
large  numbers. 

Even  with  a  low  power  the  invaded  cells  in  the  salivary 
gland  can  usually  be  detected,  as  they  present  a  granular 
appearance,  and  with  a  high  power,  oil  immersion,  the 
individual  sporozoites  can  be  made  out  unless  they  are 
too  numerous.  In  such  a  case  bv  pressure  on  the  cover- 
glass  the  cells  may  be  ruptured  and  sporozoites  will  be 
poured  out  in  a  manner  similar  to  that  in  which  they 
were  poured  out  on  rupture  of  a  mature  zygote. 

The  cells  in  the  middle  lobes  of  the  salivary  glands  are 
the  ones  which  most  frequently  contain  sporozoites,  and 
usually  cells  in  the  middle  lobes  of  both  glands  are  in- 
vaded, but  they  may  be  found  in  cells  in  any  of  the  lobes. 
Usually  when  scantv  they  are  found  in  cells  near  the  tips 
of  the  lobules. 

The  demonstration  of  the  development  of  Filaria 
nocturna  in  mosquitoes  is  even  simpler. 

In  the  first  twenty-four  hours  the  lilaria  embryos  will 
be  found  living  in  the  stomach  and  will  be  seen  to  be 
actively  locomotive  and  to  have  cast  their  sheaths  (ccdysis). 
Empty  sheaths  may  also  be  found.  Later  the  lilaria' 
will  be  found  by  teasing  out  the  muscular  masses,  after 
the  removal  of  the  stomach,  especially  those  of  the  thorax. 
Normal  saline  solution  should  be  used,  as  pure  water  is 
apt  to  destroy  the  worms. 

Every  stage  in  the  development  can  be  traced  by  dis- 
secting daily  one  or  two  of  a  number  of  mosquitoes  found 
to  carry  this  filaria  and  fed  at  night  on  a  person  harbouring 
F.  nocturna. 

F.  nocturna  has  been  shown  to  be  carried  by  several 
species  of  mosquitoes  belonging  to  several  genera  ;  Culex 
fatigans,  Mansonia  uniformis  and  albipes,  Cellia  argyro- 
tarsis,  Pyretophorus  costalis,  Myzorhynchus  sinensis  and 
barbirostris  are  amongst  these. 

So  far  experiments  with  Stegomyia  fasciata  have  always 


FILARIAL    LARVAE  257 

failed.  Occasionally  the  filariae  make  their  way  into  the 
muscles  and  become  encysted,  but  development  is  slow 
and  incomplete  and  the  larvae  die  and  become  absorbed 
without  reaching  the  full  larval  development.  Myzomyia 
funesta  does  not  carry  F.  noctuma. 

Temperature  has  an  important  influence,  and  at  low 
temperature,  even  with  a  suitable  species  of  mosquito, 
no  development  takes  place,  and  at  intermediate  tem- 
peratures development  is  much  retarded. 

The  points  to  observe  in  the  larvae  are  the  alterations 
in  size  and  shape,  the  variations  in  motility,  and  the 
formation  of  intestinal  and  other  structures. 

The  larva  as  found  at  first  in  the  muscles  is  exactly  like 
the  embryo  freshly  escaped  from  the  sheath.  It  soon 
becomes  less  actively  motile  and  thicker.  The  extreme 
tail  of  the  worm  does  not  become  thicker,  so  that  we  soon 
have  a  body  like  an  elongated  sausage  with  a  small  thin 
tail.  This  tail  retains  its  mobility  longer  than  any  other 
part  of  the  worm.  The  embryo  increases  in  length,  an 
alimentary  canal  with  a  terminal  mouth  and  subterminal 
anus  is  formed,  and  the  mobile  tail  disappears.  At  this 
stage  only  very  sluggish  occasional  movements  can  be 
observed.  The  larva  continues  to  elongate  and  again 
becomes  actively  motile.  At  this  stage  the  alimentary 
canal  is  complete,  and  there  are  three  small  projections 
developed  at  the  tip  of  the  tail. 

The  actively  motile  young  filariae  now  escape  from  the 
muscles  and  pass  towards  the  head  of  the  mosquito  and 
from  there  into  the  labium,  where  they  can  be  found 
in  pairs,  or  in  larger  numbers,  stretched  out  with  their 
heads  towards  the  tip  of  that  organ. 

It  will  be  remembered  that  the  labium  is  the  only  part 
of  the  proboscis  that  does  not  penetrate  the  skin. 

In  order  that  the  young  filariae  may  obtain  access  to 
man  it  is  therefore  necessary  that  they  must  escape  from 
the  labium  and  find  their  own  way  down  the  puncture 
made  by  the  other  elements  of  the  proboscis. 

The  most  probable  supposition  as  to  the  course  taken 

17 


250  FILARIA    IMM1TIS 

is  that  the  worms  make  their  escape    through    the  thin 

membrane  stretched  between  the  bases  of  the  labella, 
known  as  Dutton's  membrane.  This  membrane  is  the 
weakest  part  of  the  labium  and  is  put  on  the  stretch  when 
the  two  labella  are  pushed  against  the  skin  and  separated 
as  the  piercing  elements  of  the  proboscis  are  plunged  into 
the  skin.  In  the  angle  between  the  two  diverging  labella 
the  young  worms  would  readily  burst  through  this  mem- 
brane and  enter  the  skin,  passing  through  die  glandular 
ducts  as  has  been  shown  by  Fulleborn  and  others.  That 
the  filariae  escape  from  the  labium  in  this  way  was 
surmised  by  Drs.  Annett  and  Dutton,  and  Bancroft  has 
shown  that  by  pressure  on  the  proboscis  of  a  mosquito 
in  which  these  hTaria  larvae  are  present  the  larva.-  are 
extruded  between  the  labella. 

The  further  development  of  the  young  hlarue  in  man 
is  not  known.  At  the  last  stage  of  development  in  the 
mosquito  the  worms  are  not  only  small,  i'6  mm.,  but 
sexually  immature. 

Further  growth  and  impregnation  of  the  female  must 
take  place  in  man  before  embryos  are  again  formed, 
appear  in  the  blood,  and  are,  in  turn,  taken  up  by 
mosquitoes. 

It  is  obvious,  from  the  above,  that  a  patient  harbouring 
adult  filaria  and  with  embryos  in  his  blood  can  not  only 
cause  infection  of  others,  but  continued  and  repeated 
reinfection  of  himself. 

Another  filaria  known  to  be  carried  by  mosquito<  - 
F.  immitis  of  dogs.  This  is  a  sheathless  filarial  embryo, 
but  instead  of  passing  through  the  walls  of  the  stomach 
of  the  mosquito  it  passes  up  the  lumen  of  the  Malpighian 
tubes  and  there  it  further  develops  and  passes  through  its 
non-motile  stage.  When  the  larvae  again  became  motile 
they  burst  through  the  Malpighian  tubes  and  work  their 
way  through  the  tissues  of  the  mosquito  to  the  head 
and  enter  the  proboscis  just  as  the  young  /*'.  bancrofti 
does. 

The  demonstration  of  the  development  of  the  filaria  is 


BACILLI    IN    MOSQUITOES   AND    LARVAE  259 

best  done  with  fresh  specimens  of  infected  mosquitoes  by 
teasing  them  out  on  a  slide  in  normal  saline  solution. 
The  young  worm  in  the  proboscis  can  be  demonstrated 
by  breaking  across  the  proboscis.  The  various  changes 
in  the  motility  of  the  embryo  and  young  worm  can  only 
be  demonstrated  in  the  living  state. 

Sections  {vide  sections  of  mosquitoes)  are  the  best  for 
permanent  specimens,  as  the  worms  can  then  be  seen  in 
their  proper  position. 

Nothing  is  known  of  the  mode  of  development  of  the 
other  human  filaria.  Experiments  with  many  species 
of  mosquitoes  have  failed.  Other  blood-sucking  arthro- 
pods may  be  the  carriers  or  intermediate  hosts,  as  is 
known  to  be  the  case  with  filaria  of  some  of  the  lower 
animals. 

A  large  number  of  species  of  filaria  have  been  described 
in  birds.  The  intermediate  hosts  are  unknown,  and  much 
information  as  to  the  possible  methods  of  the  propagation 
of  filaria  might  be  obtained  by  systematic  experiments  on 
some  of  these  birds. 

Various  protozoa,  such  as  gregarines  and  sporozoa, 
have  been  found  in  mosquitoes  or  in  their  larvae. 

Bacilli  swarm  in  the  intestinal  tubes  of  mosquitoes ; 
they  are  particularly  abundant  in  the  air  sacs  or  diver- 
ticula from  the  upper  end  of  the  oesophagus.  Yellow 
fever  has  been  shown  to  be  carried  by  mosquitoes 
(Stegomyia  fasciata).  The  organism  of  the  disease  is  not 
known,  and  though  there  is  reason  to  believe  that  some 
development  of  the  unknown  organism  takes  place  in  the 
mosquito,  nothing  is  known  of  the  changes  that  must 
take  place. 

Dengue  Fever. — It  is  stated  that  this  disease  is  carried 
by  mosquitoes,  but  the  evidence  is  unsatisfactory. 

Mosquito  larvae  cannot  be  bred  in  sterile  water  but 
special  colour-producing  organisms  can  be  introduced 
into  the  water  in  which  the  larvae  breed  and  are  swal- 
lowed by  them.  When  pupation  occurs  the  pupae  can 
be  transferred  to  many  changes  of  sterile  water  so  that  as 
few  organisms  as  possible  are  on  the  surface  of  the  pupa. 


260  GREGARINES   IN   MOSQUITOES 

When  the  imago  emerges  in  a  sterilized  vessel  it  will  be 
found  to  contain  some  only  of  the  organisms  that  were 
present  in  the  water  in  which  the  larvae  lived,  and  others 
are  absent. 

From  this  it  appears  that  bacteria  imbibed  by  a  larva 
can  subsequently  be  distributed  by  the  adult  or  imago. 

In  this  way  Stegomyia  fasciata  is  shown  to  distribute 
Bacillus  pyocyaneus,  but  not  B.  prodigiosus  or  violaceus. 

An  old  observation  of  Ross  on  the  development  of 
certain  gregarines  in  Stegomyia  fasciata  is  an  excellent 
instance  of  the  manner  in  which  protozoa  may  be 
acquired  by  the  aquatic  larvae  and  distributed  by  the 
adult. 

These  gregarines  are  found  in  the  intestines  of  the 
young  larvae  and  pass  up  the  Malpighian  tubes.  By  the 
time  the  larvae  are  ready  to  pupate  these  gregarines  have 
become  encysted  in  the  Malpighian  tubes  and  the  cysts 
are  full  of  young  gregarines.  ' 

During  pupation  the  cysts  rupture  and  the  gregarin^ 
are  set  free  to  pass  into  the  stomach. 

When  the  imago  emerges  these  gregarines  are  in  the 
stomach  of  the  mosquito  and  are  passed  with  the  first 
excrement  deposited  by  the  mosquito. 

The  dissemination  of  protozoa  and  bacteria  by  insects 
which  in  the  larval  stage  have  such  abundant  oppor- 
tunities of  acquiring  them  is  worthy  of  very  close  investi- 
gation. 


26l 


CHAPTER    XIV. 
Eggs,  Larv^:  and   Pup.e  of  Mosquitoes. 

The  eggs  of  mosquitoes  of  different  genera  vary  greatly. 
In  most  cases  they  are  laid  on  the  surface  of  water.  A 
few  species  of  mosquitoes  will  lay  eggs  in  other  situations. 
Some  of  these,  whilst  in  captivity,  can  be  induced  to  lay 
on  many  damp  surfaces — wet  blotting  paper,  the  cut 
surfaces  of  apples,  potatoes,  and  the  like.  Grabhamia 
dorsalis,  and  some,  at  least,  of  the  Stegomyia,  lay  eggs  in 
this  manner. 

Most,  if  not  all,  of  the  species  belonging  to  the  re- 
stricted genus  Cidex  lay  their  eggs  in  masses  or  rafts. 
Other  genera  of  the  Culiciiia,  and  many  of  the  JEdince* 
form  similar  egg-rafts.  Each  individual  egg  has  its 
long  axis  vertical  to  the  surface  of  the  water,  or  nearly 
so,  and  as  the  lower  end  is  slightly  the  larger,  the  mass 
formed  by  the  aggregation  of  these  eggs  rests  with  a 
convex  surface  downwards  on  the  water  and  a  concave 
surface  upwards.  The  egg  masses,  when  first  laid,  are 
white,  but  soon  darken,  usually  to  a  black  or  dark  brown 
colour,  but  in  some  species  to  a  bright  bronze.  The 
individual  eggs  vary  according  to  species.  In  all  the 
upper  end  is  plain,  but  the  lower  may  be  plain,  spiked, 
or  ornamented  with  a  whorl.  There  is  no  definite  oper- 
culum, that  can  be  seen,  in  the  unopened  egg,  but  when 
the  larva  bursts  through,  the  eggshell  ruptures  in   a  cir- 

*  In  this  chapter  where  the  word  sEdifics  is  used  it  must  be  under- 
stood to  include  the  various  sub-families  into  which  the  old  sub- 
family /Edinoe  has  now  been  divided. 


-■'.J  EGGS   OF   MOSQ1  [TOES 

cular  manner  round  the  broad  end  of  the  egg,  and  the 
lid  thus  formed  is  pushed  aside  by  the  larva.     When  the 
hatch  the  raft  breaks  up. 
In  Anophelince  the  eggs  are  quite  different.     They  are 

never  laid  in  rafts,  but  deposited  in  little  groups  on  the 
surface  of  the  water.  After  a  time,  when  disturbed  by 
superficial  currents  in  the  water  or  in  the  air,  they  become 
scattered  and  arranged  in  patterns,  which  vary  according 
to  the  nature  and  proximity  of  the  sides  of  the  vessel,  or 
to  floating  bodies,  such  as  blades  of  grass,  pieces  of  stick, 
&C.  The  eggs  lie  horizontally  on  the  surface  of  the 
water  and  are  irregularly  spindle-shaped,  with  the  upper 
surface  flattened.  They  are  covered  with  a  thin  reticu- 
lated membrane,  which  is  closely  adherent  to  the  upper 
and  under  surfaces  and  at  the  pointed  ends,  but  is  thrown 
into  loose  folds  at  the  sides  so  as  to  form  a  projecting 
ridge  running  a  distance,  varying  according  to  species, 
towards  both  the  pointed  ends.  This  fold  is  strength- 
ened by  tranverse  thickenings,  and  air  is  contained 
between  the  folds.  The  Anophelina  egg,  therefore,  has 
on  each  side  an  air  chamber  or  float  attached,  which 
prevents  the  egg  from  sinking.  If  the  egg  does  sink,  or 
if,  when  it  has  become  adherent  to  the  sides  of  a  vessel, 
it  is  submerged,  it  does  not  hatch,  nor  does  it  if  once 
thoroughly  dried.  When  the  larva  hatches  the  eggshell 
splits  obliquely  towards  the  thicker  end,  and  pushing 
aside  the  cap  thus  formed  the  larva  makes  its  escape. 

The  eggs  of  Stegpmyia,  of  some  other  genera  of  Culicincs, 
and  of  the  Megarhinince,  are  also  laid  separately.  They 
are  oval  eggs  and  are  covered  completely  with  a  reti- 
culated membrane,  or  are  bare.  No  large  air  cells  are 
present,  but  at  first  there  is  air  in  some  of  the  small  reti- 
cular spaces.  The  eggs  may  remain  floating  and  hatch, 
but  more  frequently  sink  and  hatch  after  remaining  some 
hours,  or  even  days,  submerged.  Such  eggs  are  highly 
resistant,  and  will  withstand  prolonged  desiccation,  or 
complete  immersion  in  water. 

This    is    most    important    from   the    point    of    view    of 


EGGS   OK   MOSQUITOES 


26 


prophylaxis.      The   eggs  of    Stegomyia  fasciata    may   be 

deposited  in  shallow  puddles  at  die  end  of  a  wet  season, 
and  it'  the  puddle  dries  the  eggs  lie  in  the  dried  mud  at  the 
bottom,  and  retain  their  vitality  for  months.  With  the 
onset  of  the  next  rains,  when  the  puddle  is  re-formed, 
the  larvae  rapidly  hatch  out.  The  eggs  of  some  of  the 
mosquitoes,  e.g.,  Grabhamia  dorsalis,  with  similar  thick 
shells,  will  retain  their  vitality  all  through  the  winter. 


Fig.  114. — a,  Egg  of  Culex ;  b\  b1,  egg  of  Anopheles ;  c,  egg  of  Stegomyia  ; 
d,  egg  of  Mansonia  ;  e,  egg  of  Psorophora. 


The  species  of  Mansonia  most  frequently  observed 
rarely  lay  eggs  in  captivity.  The  eggs  are  oval,  and  pro- 
jecting from  one  end,  have  a  long  tube,  terminating  in  a 
slightly  expanded,  trumpet-shaped  opening. 

The  eggs  of  Psorophora  are  not  unlike  those  of  Sicgo- 
myia  in  shape,  but  are  rather  more  pointed.  According 
to  Dr.  W.  N.  Berkeley  they  are  "  prickly." 

Eggs  are  best  obtained  by  collecting  adult  female 
mosquitoes  and  keeping  them  in  a  small  cylindrical 
vessel,  a  wide-necked  4-oz.  bottle  is  suitable,  containing 
water.     It   is    well  to   have  some  twigs   or   fragments  of 


264  BREEDING    FROM    I.Ak'WH 

grass  Boating  on  the  water  on  which  the  mosquitoes  m  ry 
rest.  The  top  of  the  vessel  should  be  covered  with  mos- 
quito  netting   to  allow   air   to   have   free   access.     They 

should  be  fed  on  blood  as  often  as  they  will  feed. 

Larva.1  can  be  obtained  by   keeping  the  eggs  in  water 

at  suitable  temperature.  When  first  hatched  they  are 
small  and  quite  white,  but  they  soon  increase  in  size,  and 
either  in  part  or  as  a  whole  change  colour. 

They  are  voracious  and  require  abundance  of  food, 
but  with  many  species  of  mosquitoes,  particularly  with 
some  of  the  Anophelines,  the  water  must  not  be  putrid  or 
peaty. 

A  white,  flat  dish,  such  as  a  half-plate  or  full-plate 
photographic  tray,  is  as  good  a  breeding  place  as  any. 
Some  earth  should  be  placed  at  the  bottom,  and  it  is 
well  to  place  some  grass  with  the  roots  and  earth  attached 
in  two  or  three  places,  both  along  the  edge  and  also 
towards  the  middle,  so  as  to  form  at  least  one  islet.  The 
dish  should  be  filled  so  that  there  is  about  three-quarters 
of  an  inch  depth  of  water,  and  these  dishes  are  best 
prepared  a  few  days  before  the  larvae  are  placed  in  them. 
A  little  of  the  "green  slime  "  or  other  algae  found  growing 
in  fresh  water  should  be  added  and  a  few  grains  of  dry 
rice  may  be  scattered  about  the  bottom.  Abundant  food 
will  thus  be  supplied,  but  the  water  must  not  be  over- 
stocked with  vegetation,  as  if  this  decomposes  the  water 
will  be  unsuited  to  many  larvae.  Great  care  must  betaken 
that  none  of  the  natural  enemies  of  mosquito  larva.'  are 
introduced  into  the  water.  Those  most  frequently  in- 
troduced are  the  larvae  of  Agrionidce,  one  of  tin-  groups  of 
the  dragon-flies.  These  are  short,  squat,  six-legged  larvae 
with  the  characteristic  protrusiblc  prehensile  mask.  They 
are  often  introduced  with  mud  or  in  muddy  water  and 
are  most  destructive  to  other  larva.-.  Cannibal  culicid 
larvae  should  be  looked  for,  as  they  also  are  very  destruc- 
tive. They  can  be  recognized  by  the  stilt  row  of  curved 
bristles  instead  of  line  hairs  on  each  side  of  the  mouth. 

These   dishes   must  not   be  kept   in    the  dark,   must   be 


LARV/K 


26: 


well  lighted,  and  are  best  exposed  for  short  periods  to 
direct  sunlight  if  there  is  sufficient  grass  growing  to 
provide  shelter  for  the  larvae.  They  must  not  be  left 
long  enough  in  the  sunlight  to  warm  the  water. 

When  pupae  have  formed  they  must  not  be  exposed  at 
all  to  direct  sunlight. 


Fig.  115. 


Fig.  116. 


The  water  must  not  be  overstocked  with  larva?,  as 
they  are  all,  at  times,  carnivorous.  The  larvae  should 
all  be  about  the  same  age,  but  may  be  of  different  species. 
Some  large  larvas  will  destroy  the  young  of  both  their 
own  and  other  species. 

The  tops  of  the  dishes  should  be  covered  to  prevent 
the  entrance  of  dust,  a  plate  of  glass,  or  better,  a  larger 


266  COLLECTION    OF  SPECIMENS 

glass  dish  inverted  over  the  dish  containing  the  larvae, 
will  suffice. 

Larvae  can  either  be  raised  from  the  eggs  or  caught 
from  natural  waters  by  scooping  up  the  water  in  any 
receptacle.  When  large  numbers  of  larvae  are  required 
any  receptacle  from  a  bucket  downwards  will  do,  but 
where  larvae  are  scanty  they  are  best  caught  by  using 
a  dipper.  An  ordinary  white  enamelled  coffee-cup  serves 
the  purpose  well,  but  in  some  situations  a  longer  handle 
is  better  and  this  can  be  fitted  on  to  the  cup,  or  a  soup 
ladle  may  be  used. 

Some  larvae  are  most  numerous  at  the  edges  of  pools 
or  streams  in  the  shady  places.  In  using  the  dipper  the 
open  mouth  should  be  turned  towards  the  bank  and 
plunged  in,  inclined  so  that  the  water  from  the  edge 
rushes  in.  The  dipper  should,  as  soon  as  the  rush  of 
water  has  ceased,  be  turned  upright  and  removed  from 
the  pool.  (Figs.  115  and  116.)  It  should  be  allowed  to 
stand  for  a  few  minutes  till  the  mud  has  settled  and  then 
examined.  A  hand  lens  is  useful  as  the  very  young  larva? 
can  easily  be  overlooked. 

In  obtaining  the  specimens  care  must  be  taken  not  to 
disturb  the  water  in  any  way  before  using  the  dipper,  as 
larvae  readily  take  alarm  and  dart  to  the  bottom. 

In  shallow  pools  and  small  puddles,  larvae  can  be 
readily  seen  by  looking  rather  obliquely  at  the  undis- 
turbed surface  of  the  water.  When  they  occur  in  such 
situations  they  are  usually  numerous.  In  running  water 
and  in  larger  masses  of  water  they  can  rarely  be  seen  in 
this  manner,  and  unless  a  dipper  is  used  will  be  over- 
looked. In  such  situations  it  is  not  common  to  find 
them  in  large  numbers  in  any  small  surface  of  water, 
and  consequently  the  dipper  may  have  to  be  used  fre- 
quently to  demonstrate  their  presence.  Though  in  any 
small  area  of  water  examined  they  may  be  scanty  the 
total  area  of  this  class  of  breeding  place  is  so  great  that 
these  places  are  of  the  highest  practical  importance. 

Some  species  of  mosquitoes  are   more  easily  found  as 


BREEDING   PLACES  267 

larvae  because  the  adults  do  not  frequent  human  habi- 
tations. During  certain  seasons,  particularly  cold  and 
dry  seasons,  larvae  of  all  species  will  be  found  more 
readily  than  adults. 

In  making  collections  of  mosquitoes  it  is  well  both 
to  breed  from  adults  collected  in  as  many  different 
classes  of  place,  houses,  cattle-sheds,  grass  and  forest, 
as  possible,  and  also  to  rear  adults  from  larvae  or  eggs 
found  in  still  and  running  waters,  natural  pools,  small 
and  large,  and  also  in  artificial  collections  of  water. 

No  water,  even  that  in  cesspits,  is  too  foul  for  some 
species,  whilst  others  will  not  breed  in  water  that  Euro- 
peans consider  fit  to  drink. 

The  pupae  are  found  in  the  same  situations  as  the 
larvae;  they  appear  as  small  black  objects  which  are 
usually  motionless  unless  disturbed. 

The  larvae  and  pupae  can  be  transferred  from  the 
dipper  to  a  wide-mouthed  bottle  for  carriage.  Both 
larvae  and  pupae  are  easily  destroyed  if  the  water  is  kept 
in  motion,  as  they  do  not  rest  on  the  surface  sufficiently 
long  for  proper  respiration.  If  it  is  necessary  to  carry 
them  for  long  distances  it  is  well  to  make  frequent  halts 
every  half  hour  to  an  hour  and  place  the  bottle  contain- 
ing the  larvae  upright  in  a  shady  place  for  a  quarter  of  an 
hour  or  so. 

The  character  of  the  breeding  place  must  be  carefully 
noted.  The  special  points  are  :  (1)  Whether  it  is  fresh, 
or  foul,  or  brackish.  (2)  If  the  water  is  still  or  in  motion. 
(3)  Vegetation  in  the  water.  (4)  Other  larvae  or  animals 
present.  Special  attention  should  be  paid  to  animals 
that  may  prey  on  the  mosquito  larvae,  especially  fish, 
coleopterous  and  neuropterous  larvae,  &c.  (5)  Any 
special  features  either  in  the  natural  or  artificial  receptacle 
for  the  water.  (6)  Exposure  to  light,  wind,  &c,  of  the 
surface  of  the  water.  Many  of  the  Stegomyia  larvae  will 
thrive  in  darkness,  ^dine  and  Megarhinine  larvae  often 
have  peculiar  breeding  places,  such  as  the  cups  of  pitcher 
plants,  the  hollows  in   trees,  or  the   interior  of  bamboo 


268  ANATOMY    OF    I.AKV.K 

joints  or  crab-holes.  Sonic  species  will  only  he  found  in 
one  kind  of  breeding  place. 

Duration  of  larval  stage  varies  with  the  amount  ot 
food,  temperature,  and  the  species  of  the  mosquito. 
Under  the  best  conditions  of  food  and  temperature,  with 
many  mosquitoes  the  larval  stage  is  seven  or  eight  days, 
but  may  be  indefinitely  retarded  by  cold  or  insufficient 
food.  Other  mosquitoes,  under  the  most  favourable 
conditions,  require  several  weeks  for  their  development. 
Of  these,  Megarhinine  larvae  are  instances.  The  pupal 
stage  is  not  affected  by  the  food  supply,  as  the  pupa  does 
not  require  food.  It  is  prolonged  by  a  low  temperature. 
The  pupal  stage  is  about  two  clavs  with  most  species,  but 
with  Megarhinine  pupa  and  a  few  others  is  six  days. 

Anatomy  ofLarvce  andPupce. — The  larvae  vary  in  colour 
in  different  species,  but  even  in  the  same  species  varia- 
tions occur  according  to  the  degree  of  exposure  to  light 
and  the  nature  of  the  food.  In  the  more  transparent 
larvae  the  colour  of  the  intestinal  contents,  green  or 
brown,  is  more  obvious  than  that  of  the  larva  itself. 

The  larvae  of  the  Culicidce  conform  to  a  general  type. 
The  head  is  joined  to  the  thorax  by  a  narrow  neck.  In 
the  head  are  a  pair  of  compound  eyes  and  two  simple 
eyes  or  ocelli.  There  are  a  pair  of  short  antennae  and  a 
mouth  composed  of  an  upper  lip,  a  pair  of  mandible--,  a 
pair  of  maxillae,  and  a  lower  lip  or  labial  plate. 

The  thorax  is  composed  of  three  fused  segments.  There 
are  no  ambulatory  legs,  but  sensitive  and  balancing  hairs 
are  abundantly  supplied. 

The  abdomen  is  long,  and  composed  of  nine  segments. 
The  last  is  smaller,  and  inclined  at  an  oblique  angle  down- 
wards. At  the  termination  of  this  segment  is  the  opening 
of  the  anus,  surrounded  by  four  retractile  papillae,  prob- 
ably respiratory  in  function — anal  gills  or  branchiae. 

On  the  upper  surface  of  the  eighth  segment  are  the 
spiracles  or  openings  of  the  two  respiratory  tubes,  which 
run  the  whole  length  of  the  body,  and  supply  the  larvae 
with  air.      In  CorethrincE  these  tubes  are  smaller,  but  have 


ANATOMY    OF    LARV.K  269 

dilatations  on  them  in  the  thorax  and  abdomen — air- 
bladders. 

The  openings  of  these  tubes  are  direct  in  the  Anophelince, 
which  can  therefore  be  described  as  asyphonate.  In  the 
other  sub-families  the  tubes  are  continued  into  a  conical 
tube  jointed  on  to  the  upper  surface  of  the  eighth  segment, 
and  at  the  apex  of  this  tube  are  the  external  openings  of 
the  respiratory  tubes.  This  projection  is  known  as  the 
respiratory  syphon,  and  the  larva?  of  all  the  Culicidce,  except 
the  Anophelina,  are  therefore  said  to  be  syphonatc. 

The  head  is  composed  of  many  chitinous  plates,  which 
are  thicker  and  darker  on  the  dorsal  surface. 

The  opening  of  the  mouth  is  directed  slightly  down- 
wards in  all,  and  almost  directly  downwards  in  many. 

The  upper  lip  consists  of  a  middle  portion  or  palate 
supported  on  either  side  by  lateral  plates  covered  with 
bristles. 

The  pair  of  mandibles  are  placed  beneath  the  upper 
lip,  and  are  usually  toothed.     The  movements  are  lateral. 

The  pair  of  maxillae  are  below  and  behind  the  man- 
dibles ;  their  movements  are  obliquely  upwards  and 
inwards. 

The  inferior  lip,  or  labium,  is  a  triangular  plate,  usually 
very  dark  in  colour,  and  with  a  more  or  less  serrated  edge. 

On  each  side  of  the  mouth  are  chitinous  plates  attached 
to  the  mandibles  and  maxillae,  and  from  these  arise  the 
"  brushes"  or  masses  of  long,  stiff  hairs,  fine  in  most  of 
the  Culicidce,  but  thick  and  curved  in  the  larvivorous 
larvae,  which  are  so  arranged  that  they  can  be  moved 
laterally  and  folded  completely  over  the  mouth  or  thrown 
back  so  as  to  form  a  very  oblique  angle  with  each  other 
(fig.  117).  These  brushes  in  life  are  in  constant  movement, 
and  cause  a  sufficient  current  in  the  water  to  wash  solid 
suspended  particles  to  the  open  mouth  of  the  larva. 

There  are  great  variations  in  the  different  parts  which 
are  of  use  in  distinguishing  larvae  of  one  mosquito  from 
those  of  another.  Much  attention  has  been  paid  to  the 
shape  of  the  inferior  lip  plate,  which  is  a  conspicuous 
object,  and  varies  in  closely  related  species. 


270 


AXATOMY    OF    LAKV.K 


The  antennae  are  articulated  to  the  head.  They  move 
slightly.  They  are  not  truly  jointed,  but  in  some  there 
is  an  abrupt  variation  in  thickness,  probably  indicating  a 
joint.  They  vary  in  length  and  in  the  number  and 
arrangement  of  the  hairs  and  spines  ornamenting  them. 
They  are  of  value  in  differentiating  the  larvas  of  different 
species. 


>mMkM± 


FlG.  1 17- — Head  of  mosquito  larva,     a,  Mandible;   b,  lower  lip  ;  c,  antenna; 
d,  eye;  e,  brushes  ;  f,  upper  lip  ;  g,  maxilla. 


The  head  is  very  mobile  in  some  species,  and  in  the 
Anophelince  so  much  so  that  they  can  turn  the  ventral 
surface  upwards  and  feed  in  that  position.  As  the  ventral 
surface  is  light,  and  the  dorsal  black,  a  larva  may  at  one 
moment  appear  to  have  a  dark  head,  and  at  the  next  a 
light  one. 

The  thorax  is  well  supplied  with  simple  or  compound 
hairs.  The  longer  and  more  conspicuous  are  arranged 
on   each   side.     In  a   few  species  there   are    in  addition 


LARVAE  271 

strong  curved  spines  slightly  below  the  lateral  hairs.  One 
or  two  pairs  of  these  spines  may  be  present. 

On  the  abdomen  are  also  hairs,  varying  greatly  in  genera 
and  species.  In  the  Auophelines  are  peculiar  palmate 
hairs,  the  shape  of  which  varies  in  different  species. 

In  many  of  the  ALdiuce  compound  hairs  are  very 
numerous.  In  the  Stegomyia  hairs  are  scanty  and 
inconspicuous. 

The  shape  of  the  respiratory  syphon  attached  to  the 
eighth  segment  in  all  but  the  Anophelines  is  of  great 
importance.  It  varies  in  length,  so  much  so  that  attempts 
have  been  made  to  classify  the  Culicidce  on  the  "syphonic 
index,"  or  the  relative  length  of  the  syphon.  Generally 
speaking,  all  the  Megarhina,  all  larvivorous  larvae,  Stego- 
myia and  Desvbidea,  as  well  as  some  of  the  ALdiute,  such 
as  Uranotamia  and  many  other  genera,  have  short  respira- 
tory syphons.  Most  species  of  Culex,  in  the  restricted  sense, 
and  many  others  of  the  Culicince  and  JEdincz  have  long 
respiratory  syphons.  A  re-classification  on  the  basis  of 
the  syphonic  index  would  break  up  the  present  classifica- 
tions founded  on  adult  characters,  whether  those  adult 
characters  were  on  the  character  of  the  palps,  proboscis, 
or  scales.  It  would  be  less  convenient  than  the 
classification  founded   on  adult  characteristics. 

The  openings  of 'the  respiratory  tubes  at  the  end  of  the 
syphon  are  often  guarded  by  mobile  flaps,  and  hairs  and 
spines  are  present  on  the  syphons  in  most  species.  In 
some  of  the  JEdinaz  these  are  very  numerous. 

The  alimentary  system  of  the  larva  consists  of  a  tube, 
apparently  structureless  and  of  uniform  calibre,  running 
from  the  mouth  to  the  anus.  In  the  more  fully  grown 
specimens  this  tube  is  seen  to  be  contained  inside  the  true 
intestine,  which  is  arranged  as  in  the  adult.  In  the  space 
between  is  clear  fluid  not  containing  any  food  particles. 
The  Malpighian  tubes  and  other  appendages  of  the 
alimentary  canal  of  the  adult  are  present  at  this  stage. 

The  intestinal  system,  including  the  inner  tube  con- 
taining the  food,  and  the  outer  tube  and  appendages,  can 


272  EGGS,    LAKV.K    AND    I'lI'.K 

be  pulled  out  of  the  larval  case  in  a  manner  similar  to 
that  by  which  the  intestine  is  removed  from  the  adult, 
but  it  is  more  easily  done  by  extraction  through  the 
anterior  part  of  the  larva  than  from  the  tail. 

The  space  between  the  temporary  and  permanent 
intestine  may  contain  gregarines  and  numerous  micro- 
organisms, and  it  is  to  this  space  that  attention  should  be 
paid  in  investigating  the  conveyance  of  parasites  acquired 
by  larvae. 

The  respiratory  system  of  the  larva  is  comparatively 
simple.  At  the  end  of  the  respiratory  syphon,  if  there 
be  one,  or  from  the  dorsal  surface  of  the  eighth  abdominal 
segment,  are  the  openings  leading  into  the  two  main 
trachea?,  which  pass  up  the  abdomen,  giving  off  branches 
to  each  segment  and  inosculating  freely  in  the  thorax. 
They  send  off  branches  to  the  various  parts  here  and  to 
the  head. 

The  Pupa. — When  the  larva  has  reached  its  full  stage 
of  development  the  thorax  becomes  swollen,  the  cuticle 
with  all  the  appendages  is  detached  and  cast  off  and  the 
larva  becomes  a  pupa.     The  organs  are  already  formed. 

The  pupa  differs  most  materially  from  the  larva  in 
that  there  is  no  longer  a  mouth  opening  externally, 
and  the  respiration  is  conducted  through  two  tubular 
openings  arising  on  each  side  of  the  compound  head 
and  thorax.  The  change  in  appearance  is  great,  the 
head  and  thorax  are  fused,  and  the  only  external  append- 
ages are  the  two  respiratory  tubes.  The  abdomen  is  still 
segmented  and  is  usually  curved,  so  that  the  termination 
is  under  the  compound  thorax.  It  terminates  in  two 
large  fins. 

The  pupal  stage  is  a  comparatively  short  one.  There 
is  no  possibility  of  feeding  and  the  pupa  remains  quiet, 
breathing  through  the  respiratory  tubes  unless  disturbed, 
whilst  the  more  complete  development  of  the  imago 
takes  place  within  its  sheath.  The  duration  of  the  pupal 
stage  is  affected  by  the  temperature,  but  is  usually  from 
two  to  five  days.     The  pupae    of   some  species  will  not 


LARWE  273 

remain  alive  longer  than  a  tew  days  if  the  conditions  are 
not  favourable  for  development. 

In  the  examination  of  eggs,  larvae  and  pupa?,  the  points 
to  be  observed  are  as  follows  : — 

Eggs. — (1)  The  size,  shape,  colour.  (2)  The  manner 
in  which  the  eggs  are  arranged  and  where  deposited. 
(3)  The  character  of  any  thickenings  or  other  external 
markings.  (4)  The  length  of  time  required  under  stated 
conditions,  temperature  and  so  on,  between  the  deposi- 
tion of  the  eggs  and  the  hatching  of  the  larvae,  and  any 
variations  noted  with  variations  of  conditions.  (5)  The 
effect  of  desiccation,  immersion  and  temperature  on  the 
vitality  of  the  eggs. 

Larva. — In  the  larva  the  relative  sizes  and  shapes  of 
the  different  divisions — head,  thorax  and  abdomen.  The 
character  of  the  head  appendages,  the  antennae,  mouth 
apparatus,  &c.  Any  marked  colouring.  Much  work  has 
been  done  on  the  differences  in  the  appendages  of  the 
head  of  Anophcliiia  larvae,  and  it  has  been  shown  that  the 
differences  are  so  marked  in  their  arrangement  that  many 
of  the  species  can  be  distinguished  as  larvae. 

In  the  thorax  the  character  of  the  lateral  hairs  and  any 
characteristic  markings  must  be  noted. 

In  the  abdomen  the  points  of  greatest  importance  are 
the  appendages  on  the  eighth  and  ninth  segments.  The 
presence  or  absence  of  a  respiratory  syphon  attached  to 
the  eighth  segment  is  one  of  the  most  important  generic 
differences.  Where  present  it  varies  in  length  and  shape 
in  different  genera.  In  different  species  it  varies  in  colour 
and  in  the  distribution  of  colour  so  markedly  that  it  is 
often  easier  to  distinguish  between  different  species  by 
the  character  of  the  syphon  than  it  is  to  distinguish  be- 
tween the  adults.  Varying  positions  of  larvae  are  associ- 
ated with  the  differences  in  length  or  the  absence  of  the 
syphon. 

The  arrangement  of  bristles  and  hairs  on  the  eighth 
and  ninth  segments  presents  marked  differences  in  the 
different  species.  In  Anophcliiia  on  the  other  segments, 
18 


274  I.AK'V.K 

in  addition  to  the  lateral  hairs,  there  is  on  each  side  a  row 
of  stellate  or  palmate  hairs.     These  are  nearer  the  middle 

line  than  the  simple  bristles,  and  the  stellate  portion  forms 
a  kind  of  cup.  This  adheres  to  the  surface  film  of  the 
water  and  aids  the  larva  in  maintaining  its  horizontal 
position. 

Colouring  of  larvae  is  of  less  importance,  as  in  some 
species  the  colour  may  vary  from  yellow  to  green,  brown, 
or  even  black.  In  others  variations  are  comparatively 
small,  these  are  usually  dark  under  all  circumstances. 

In  noting  the  colour  any  conspicuous  marking  must 
be  mentioned,  the  conditions  under  which  the  larvae  were 
grown,  and  whether  or  not  change  of  conditions,  such 
as  greater  light,  different,  food,  &c,  results  in  a  change  of 
colour. 

The  nature  of  the  food  can  be  determined  by  the 
examination  of  the  contents  of  the  intestine,  or  by  watch- 
ing the  larvae  feed  in  water  containing  a  mixture  ot 
natural  foods.  It  will  be  found  to  vary.  The  kind  of 
food  on  which  they  thrive  best  should  be  noted. 

The  duration  of  the  larval  stage  under  as  many  divers 
conditions  as  possible,  including  exposure  to  light,  heat, 
and  cold,  and  any  observations  as  to  the  conditions  pre- 
disposing to  death  or  leading  to  an  undue  proportion  of 
males  in  the  imagines,  should  be  noted. 

In  breeding  from  larvae  it  is  most  important  that  the 
water  should  be  properly  oxygenated.  Darling  advise- 
that  a  jet  of  air  should  be  passed  through  by  means  of 
a  Pacquelin  cautery  bulb,  having  a  heavy  glass  perforated 
tip,  once  or  twice  a  day. 

The  important  natural  enemies  of  the  mosquito  larvae 
are  fish,  larvie  of  other  insects,  particularly  those  of  the 
dragon-fly,  &c.  Where  possible  the  species  of  these 
enemies  should  be  determined.  If  the  larvae  are  caught 
as  larva.-  and  not  reared  from  eggs  particular  care  should 
be  taken  to  observe  the  nature  of  the  places  in  which 
they  were  found. 

Breeding    places    of    the    known     carriers    of    disease, 


BREEDING   PLACES  275 

such  as  Anophelince,  Stegomyia  and  L'ulcx,  require  very 
detailed  consideration.  It  is  convenient  to  divide  these 
into  permanent  waters  such  as  will  withstand  a  con- 
siderable period  of  rainless  weather,  and  temporary  waters, 
which  require  frequent  renewal.  r\  ney  may  be  natural 
or  artificial.  Of  permanent  waters,  rivers,  large  ponds 
and  the  edges  of  lakes  under  certain  conditions  are  of 
the  utmost  importance.  In  such  situations  the  larvae 
are  usually  widely  scattered,  and  without  the  repeated 
routine  use  of  a  dipper  such  places,  often  the  most  im- 
portant, are  usually  overlooked. 

The  conditions  favourable  are  the  growth  of  grasses, 
reeds  or  sedges  in  the  water.  These  growths  check  the 
stream,  provide  food,  and  protect  to  some  extent  the 
larvae  from  their  natural  enemies. 

There  are  two  main  classes  of  growths  important  : — 

(1)  Those  growing  from  the  bed  of  the  river  or  lake, 
in  the  shallows  and  on  shelving  banks.  The  height  of 
the  water  greatly  affects  the  area  suitable.  The  taller 
and  thicker  sedges  are  not  so  suitable  as  the  lower  and 
thinner  ones,  probably  on  account  of  the  absence  of 
light  and  too  great  stagnation  of  the  water  (fig.  115). 

(2)  Those  growing  from  floating  masses  of  roots  and 
attached  to  the  earth  only  near  the  edge  of  the  river. 
The  raft  formed  by  the  closely  interlaced  roots  is  sub- 
merged by  the  weight  of  the  grass  growing  in  the  air,  and 
in  the  shallow  water  lying  above  this  raft  of  roots  Ano- 
pheline  larvae  breed  freely.  No  alteration  in  the  level  of 
the  water  makes  any  material  difference  to  this,  a  com- 
mon class  of  breeding  place  (fig.  119).  In  flood  times 
islets  of  this  floating  grass  are  torn  off  and  carried  down 
the  stream,  carrying  with  them  larvae,  and  in  this  manner 
they  may  be  carried  long  distances  down  the  river.  It 
is  not  improbable  that  the  cutting  of  the  sudd  in  rivers 
may  result  in  larvae  of  mosquitoes  being  carried  for  long 
distances  clown  the  river  and  thus  extending  the  area  of 
distribution  of  these  mosquitoes,  but  according  to  Balfour 
no  such  result  seems  to  have  taken  place  on  the  Nile. 


2/6 


BREEDING    PLACES 


Rivers  are  dangerous  when  variations  in  level  are  not 
too  great  or  too  rapid.  Such  streams  as  have  a  constant 
supply  independent  directly  of  the  rainfall  are  particu- 
larly dangerous.  Such  sources  are  the  melting  of  the 
snow  from  snow-covered  mountains  and  the  effluents 
of  large  lakes. 

Springs  which  often  arise  on  the  slopes  of  lulls  are  other 
important  permanent  breeding  places.  These  usually 
commence  as  a  small  pool  with  a  surrounding  swampy 


Fig.   118. 


Fig.   119. 


area.  The  grasses  round  are  often  of  different  species  or 
grow  more  luxuriantly  than  elsewhere,  and  these  places 
can  therefore  usually  be  identified  with  ease. 

The  streams  arising  from  such  springs  are  not  of  much 
importance  during  heavy  rains,  but  when  the  water 
supply  is  diminished,  wherever  the  streams  spread  into 
swampy  areas,  or  form  pools  fringed  with  vegetation,  or 
in  backwaters,  larva3  are  usually  to  be  found  with  the  aid 
of  the  dipper.  In  some  of  these  situations  they  are  carried 
by  the  streams  from  the  springs  or  other  breeding  places. 
In  others  the  eggs  may  be  deposited  and  hatch  in  the 
place  in  which  the  larvae  are  found.     Amongst  the  easiest 


BREEDING    PLACES  277 

places  to  find  larva?  are  the  pools  left  in  the  bed  of  such 
a  stream  when  the  spring  commences  to  dry  up,  par- 
ticularly if  a  small  current  connects  the  pools  and  keeps 
the  water  fresh.  Some  springs  will  dry  up  after  a  month's 
dry  weather,  others  in  three  or  four  months,  but  some 
are  usually  permanent,  though  the  water  may  be  scanty, 
from  one  wet  season  to  another. 

Swamps,  unless  kept  supplied  by  fresh  water,  are  not 
suitable  breeding  places  for  many  species.  For  other 
species  they  are  suitable,  provided  the  vegetation  is  not 
too  rank. 

A  high-level  subsoil  water  may  lead  to  formation  of 
natural  permanent  pools.  On  the  sandy  shores  of  great 
lakes  the  sand  is  usually  thrown  up  into  a  ridge  with  a 
hollow  behind  it,  and  in  this  hollow,  as  long  as  the  lake 
level  is  high,  water  will  be  present  and  forms  a  suitable 
breeding  place.  As  the  lake  level  reaches  its  greatest 
height  at  the  end  of  the  wet  season  and  very  slowly 
falls  in  the  dry  season,  these  pools  may  persist  in  the 
vicinity  of  such  lakes  for  some  months  after  the  rains 
have  ceased. 

Temporary  breeding  places  are  of  many  different  classes. 
Almost  any  hollow  or  hole  that  will  contain  water  is  a 
suitable  breeding  place  during  continuous  rains.  If  the 
rain  be  intermittent,  only  such  places  as  can  retain  water 
during  the  periods  of  intermission  are  suitable.  Such 
places  require  a  frequent  and  heavy  rainfall  and  an  im- 
pervious soil,  and  are  not  often  found  except  under  these 
conditions.  The  "Anopheles'  pool"  most  often  described 
belongs  to  this  class,  and  is  exceptional  in  many  places 
where  Anophelincc  are  abundant. 

A  place  that  is  frequently  flushed  is  not  a  suitable 
breeding  ground,  but  irrigation  trenches  or  natural 
hollows  are  good  breeding  places  for  some  species  if  the 
area  of  the  trenches  is  such  that  the  water  supplying  it  is 
insufficient  to  flush  it  in  its  whole  extent. 

Artificial  Breeding  Places. — Borrow  pits  at  the  sides  of 
railway   embankments,    the    trenches   so    often    made    in 


278  ARTIFICIAL   BREEDING    PLACES 

the  course  of  road-making,  and  hollows  or  furrows  made 
in  native  or  other  gardens,  are  common  breeding  places 
of  Anophelines  and  some  other  mosquitoes.  A  high  level 
of  the  subsoil  water  is  necessary  for  these  places  to  be  ol 

importance. 

Irrigation  systems  where  the  water  supply  is  con- 
tinuous but  insufficient  to  Hush  are  important  places.  In 
any  case,  even  with  a  well-designed  system,  if  the  source 
of  the  water  be  from  a  natural  breeding  place  larvae  will 
be  conveyed  all  over  the  irrigation  system. 

Instances  occur  in  which  larvae  are  conveyed  for  over 
a  mile  by  such  a  trench  from  a  natural  permanent  breed- 
ing place  to  a  European  settlement. 

Obstructions  in  the  course  of  a  stream,  such  as  Irish 
crossings,  dams,  &c,  may  convert  an  inferior  natural 
breeding  ground  into  an  excellent  one. 

Badly  graded  gutters,  broken  bottles,  water-butts,  empty 
tins  and  any  artificial  receptacle  that  will  hold  water  are 
preferential  breeding  places  for  some  species  of  mos- 
quitoes, particularly  those  belonging  to  the  genus 
Stegomyia. 

Wells  in  many  places  do  not  seem  to  be  breeding 
places,  but  in  other  places  they  certainly  are. 

On  the  whole,  artificial'  breeding  places  are  usually  the 
work  of  Europeans,  and  the  worker  in  the  Tropics  has 
rarely  to  go  beyond  his  own  grounds  to  find  larvae  of 
several  species  of  mosquitoes. 

Too  little  attention  has  been  paid  to  the  breeding  places 
of  different  species.  We  know  that  great  differences 
occur  in  the  preferential  breeding  places  of  different 
species  as  of  different  genera,  but  little  exact  work  has 
been   done  on   the  subject. 

For  exact  descriptions  of  the  larvae  of  one  species  which 
might  serve  as  an  example  the  reader  is  referred  to  the 
articles  on  Anopliclcs  maculipennis,  by  Nuttall  and  others, 
in  the  first  volume  of  the  Journal  of  Hygiene. 

The  pupce  differ  less  from  each  other  than  the  larvae, 
and  many  insects  form  pupae  that  are  not  unlike  those  ol 


CARRIAGE   OF   MOSQUITOES  2/Q 

the  Culicidce.     The  greatest  differences  arc  to  he  observed 

in  the  respiratory  tubes.  In  all  the  Culicidce  they  are- 
simple  tubes  with  one  opening.  In  the  Anopheles  the 
opening  of  the  tube  is  a  wide,  expanded,  trumpet-shaped 
one;  in  the  Culex  the  opening  is  more  of  a  slit  and  the 
termination  is  little  expanded.  In  Mansonia,  according 
to  Low,  the  tubes  are  very  long  and  slightly  bent  for- 
ward. In  the  different  species  there  are  variations  in  the 
size  of  the  pupa  and  in  the  colour.  The  majority  are, 
after  exposure  to  light,  brown  or  black,  though  when  first 
formed  they  are  yellow.  A  few  are  green,  though  most 
of  these  become  dark  before  maturity. 

To  hatch  out  the  pupae  all  that  is  required  is  that  they 
should  not  be  disturbed  and  that  they  should  be  kept 
in  clean  water.  No  food  is  needed.  They  should  be 
kept  in  a  half  light. 

Carriage  of  Mosquitoes. — Mosquitoes  may  be  carried 
in  any  stage  of  their  existence,  As  eggs  they  are  not 
very  easy  to  carry,  as  those  that  float  are  often  washed 
on  to  the  sides  of  the  vessel  and  there  are  dried  and 
killed.  Eggs  like  those  of  Stcgomyia,  which  sink  and 
are  not  injured  by  immersion,  are  easily  carried,  and  it 
is  probably  owing  to  this  that  these  mosquitoes  or  their 
larvae  are  so  often  found  on  board  ship. 

The  carriage  of  larvae  we  have  already  dealt  with. 
For  the  development  of  many  species  light  is  a  necessity, 
and  consequently  such  species,  including  most  of  the 
Anophcliiuv,  are  not  carried  far  on  board  ship,  as  most  of 
the  fresh  water  is  necessarily  in  closed  casks  or  other 
dark  receptacles. 

The  adult  mosquitoes  must  be  carefully  carried  as 
they  are  easily  injured  by  rough  handling  or  bruising. 
On  the  whole  glass  vessels  should  be  avoided  because 
of  the  hard  surface  of  the  glass.  Mosquitoes  cannot 
hold  on  to  it.  If  fresh  grass  or  other  moist  substances 
be  placed  in  the  glass  vessel,  water  of  condensation  is 
often  deposited  on  the  glass,  and  the  mosquitoes  adhere 
by  the  wings  to  this  wet  surface  and  speedily  die. 


;8o 


CARRIAGE   OF   MOSQUITOES 


If  glass  vessels,  test  tubes,  &c,  are  used,  the  mosquitoes 
must  be  carried  very  carefully,  and  no  water  be  placed 
in  the  vessel.  Jungle  mosquitoes  will  be  killed  by  ex- 
posure to  dry  air,  and  with  them  the  plug  of  cotton-wool 
should  be  kept  wet. 

A  light  cage  covered  with  mosquito  netting  is  as  good 
an   arrangement  as  any,  though  at  a  pinch  a  small  box 


Fig.   i 20. 


covered  with   netting  on  the  open   side  will   work  satis- 
factorily. 

The  box  designed  by  Dr.  Sambon  and  containing  four 
compartments,  each  containing  a  cylindrical  wire  cage 
covered  with  netting,  is  an  excellent  one  (rig.  120).  It 
was  in  such  cages  that  infected  mosquitoes  were  sent 
from  Italy  to  the  London  School  of  Tropical  Medicine 
for  the  well-known  infection  experiments,  which   resulted 


MOSQUITO   CAGES 


281 


in  the  practical  demonstration  that  mosquitoes  infected 
with  the  malaria  parasite  could  infect  men  in  a  country 
where  there  was  no  other  possibility  of  acquiring  an 
infection. 

A  simple  cage  can  be  made  by  having  a  portable  wire 
cage  that  can  be  folded  flat,  and  bags  of  mosquito  netting 
which  are  pulled  over  the  framework.  The  open  end  is 
then  tied  in  a  knot  (fig.  121). 


Fig.   121. 


Adult  mosquitoes  can  be  kept  in  test  tubes  or  wide- 
necked  bottles  covered  with  fine  gauze  or  mosquito 
netting.  A  little  water  should  be  placed  at  the  bottom 
and  some  resting  place,  such  as  a  piece  of  stick,  blade  of 
grass,  or  folded  card  placed  above  the  water. 

Many  mosquitoes  will  feed  readily  through  the  netting, 
others  will  not,  though  they  feed  readily  in  a  larger  space. 

Mosquitoes  thrive  better  if  kept  in  a  larger  space. 
The  box  slightly  modified  from  Dr.  Sambon's  cage  is 
very  convenient  for  this  purpose.  The  front  is  com- 
posed of  glass  in  two  pieces  for  convenience  in  packing, 


282 


MOSQUITO   CAf.l.s 


while  the  ends  are  of  line  wire  gauze  to  allow  the  entrance 
of  air.  The  hole  in  the  centre  of  this  gauze  is  covered 
by  cotton  sleeves.  These  are  convenient  for  the  intro- 
duction of  an  arm  for  feeding  experiments.  Through 
these  holes  the  hand  and  test  tube  can  be  introduced 
when  we  wish  to  catch  a  mosquito.  The  pieces  of  gl  tss 
forming  the  face  of  the  box  slide  in  a  groove,  and  can 
be  removed  when  required  (hg.  122). 


Fig.   122. 

Ripe  fruit,  such  as  apples,  dates,  and  bananas,  serves  as 
food  for  mosquitoes,  but  some  will  not  lav  eggs  unless 
supplied  with  blood.  As  substitutes  for  fruit,  sugar, 
syrups  or  jams  will  serve.  Some  species,  Stegomyia 
calopus  and  Culex  fatigans,  for  example,  are  easy  to  keep 
in  captivity,  and  can  be  kept  alive  for  months  ;  others 
will  die  in  a  few  days. 

Ants  of  many  kinds  are  very  destructive  to  mosquitoes, 
particularly  to  those  confined  in  small  spaces.  To  avoid 
this  the  mosquito  cage  should  be  placed  on  legs,  each  of 
which  rests  in  a  small  tin  containing  kerosene. 


283 


CHAPTER    XV. 

Fleas,  Lice  and   Bed-bugs. 

In  view  of  the  evidence  that  rats  and  their  parasitic 
insects  play  an  important  part  in  the  dissemination  of 
plague,  it  is  considered  desirable  to  include  here  some 
notes  on  fleas  that  may  aid  the  worker  in  recognizing 
the  more  common  species. 

It  has  long  been  known  that  fleas  may  serve  as  the 
intermediate  hosts  of  certain  parasites.  The  cysticercus 
stage  of  Dipylidium  caninum  is  passed  in  the  dog-flea, 
Ctenocephalus  serraticeps,  as  well  as  in  the  dog-louse,  and 
healthy  rats  have  been  infected  with  Trypanosoma  lewisi 
by  allowing  fleas  taken  from  infected  rats  to  feed  upon 
them.  The  work  of  the  Indian  Plague  Commission,  as 
well  as  observations  elsewhere,  show  that  fleas  are  the 
carriers  of  the  ■"•Bacillus  pest  is  from  rats  to  rats  and  also 
from  infected  rats  to  man  and  other  animals. 

Fleas  are  usually  included  in  the  group  Insecta  as  an 
order,  the  Siphonaptcra  (vide  p.  157).  They  differ  from 
most  dipterous  insects,  not  only  in  the  absence  of  wings 
but  also  in  that  the  three  thoracic  segments  are  distinct 
from  one  another,  and  that,  beyond  carrying  legs,  these 
segments  differ  but  little  from  the  abdominal  segments. 
The  antennae  also  differ  from  those  found  in  Diptera. 

Capture  and  Examination  of  Fleas. — To  collect  fleas 
from  rats,  mice,  &c,  it  is  best  to  confine  the  animal  in 
a  vessel  at  the  bottom  of  which  is  a  piece  of  cotton-wool 
soaked  in  chloroform.  The  fleas  are  killed  more  quickly 
than  the  host,  and  may  be  picked  from  among  the  hairs 
or  from  the  bottom  of  the  vessel.     If   it  is  required  to 


284 


I-  I.I  As 


examine  the  internal  structure  this  method  is  not  very 
satisfactory,  as  the  fleas  are  frequently  found  filled  with 
blood,  and  this  obseures  details.  A  preferable  method  is 
to  capture  them  alive  and  keep  them  in  a  test-tube  until 
they  die  of  starvation. 

They  may  be  examined  directly,  or  if  it  is  desired  to 
mike   permanent  preparations  they  should  be   rendered 


Lp      Md  K  Md    Lp. 


/-Mx.  p. 


— Mx. 


Fig.  123. — Mouth-parts  of  a  Plea  (Veri/u'psyl/a  a/akurt,  i).  (Afier 
Wagner.)  &.,  Median  lancet;  lp.,  labial  jialpi  ;  md.,  mandibles;  mx., 
maxillae;  mx.p.,  maxillary  palpi. 


transparent  and  mounted.  There  are  many  methods  for 
clearing  and  mounting  small  insects.  Glycerine  is  a  good 
clarifying  medium,  but  it  takes  a  long  time  to  penetrate. 
A  method  that  gives  good  results  is  to  fix  in  absolute 
alcohol,  leaving  them  about  one  day  to  completely 
dehydrate,  clear  in  xylol  and  mount  in  Canada  balsam. 
Some  authorities  use  turpentine  or  creasote  for  clearing. 
The  preparations  should  be  examined  not  only  by  trans- 
mitted light,  but  also  by  direct  illumination  to  note  the 
depressions,  the  direction  of  hairs,  &c. 

External   Anatomy. — Mouth-parts. — These    comprise    a 


EXTERNAL   ANATOMY  285 

perforating  and  suctorial  tube  and  two  free  pieces,  the 
maxilla;  (fig.  123). 

The  max illce  (mx.)  have  the  form  of  a  triangular 
pyramid,  the  apex  of  which  projects  downwards.  Arising 
from  the  base  of  each  maxilla  is  the  maxillary  palp 
(mx.p.). 

The  piercing  and  suctorial  apparatus  is  formed  by  the 
mandibles  (rad.)  and  the  epipharynx  (h.).  The  labial 
palps  (l.p.)  act  as  sheaths. 

The  mandibles  have  the  form  of  elongated  styles  with 
serrations  along  their  distal  two-thirds.  Each  contains 
on  its  mesial  surface  a  salivary  groove,  which  at  the  base 
widens  out  into  a  trough  and  towards  the  tip  is  nearly 
closed  by  the  approximation  of  its  edges  forming  a  canal. 

The  epipharynx  is  a  pointed,  pricking  organ,  grooved 
ventrally.  By  the  approximation  of  the  epipharynx  and 
the  two  mandibles  a  channel  is  formed  along  which 
blood  is  sucked. 

The  epipharynx  makes  a  way  through  the  skin,  and 
the  mandibles  enlarge  and  lacerate  the  hole  thus  made, 
and  convey  into  it  the  salivary  secretion,  and  blood  is 
aspirated  from  the  wound  along  the  channel  formed  by 
the  epipharynx  and  mandibles. 

The  anlennce  are  contained  in  fossae  at  the  back  part 
of  the  head,  behind  the  eyes  when  these  are  present,  and 
are  directed  obliquely  downwards  and  backwards.  It 
is  important  not  to  mistake  the  maxillary  palps  for  them. 
The  antennas  are  three-jointed,  the  third  joint  being 
ringed. 

The  three  thoracic  segments  known  as  pro-,  meso-, 
and  meta-thorax  are  freely  movable  on  one  another, 
and  to  each  segment  is  attached  a  pair  of  legs.  Each 
leg  is  described  as  composed  of  five  pieces — coxa,  tro- 
chanter, femur,  tibia,  and  tarsus,  the  latter  consisting 
of  five  segments,  to  the  last  of  which  are  attached  two 
claws.  The  hairs  on  the  last  joint  of  the  hindmost 
pair  of  legs  are  of  value  in  classification. 

The  abdomen  is  oval  in  shape  and  is  composed  of  nine 


286 


FLEAS — METAMORPHOSIS 


segments.      The  ninth   segment   is   the   smallest   and   is 
known  as  the  pygidium. 

The  males  arc  recognized  by  their  small  size  and  by 
the  presence  of  the  coiled  penis  extending  sonic  distance 
inside  the  abdomen. 

Metamorphosis  of  Fleas. — The  metamorphosis  in  Ilea-, 
is  complete.  The  eggs  are  laid  at  all  seasons  of  the  year, 
but  their  development  is  more  rapid  in  summer  than  in 
winter.  The  female  does  not  attach  the  eggs  to  the  skm 
or  hairs   of  the  host,  but  allows  them  to  fall   anywhere, 


Fig.  124.— a,  Head  ;  B,  thorax,  (1)  proihorax  or  pronotum,  (2)  mesothorax 
or  mesonotum,  (3)  metathorax  or  metanotum  ;  c,  abdomen. 


whether  it  be  on  the  host,  on  the  earth,  or  elsewhere. 
The  ova  are  small,  ovoid  or  rounded  in  shape,  and 
usually  eight  to  twelve  are  laid.  The  larva  is  hatched 
out  in  four  to  six  clays  in  summer  and  in  nine  to  twelve 
days  in  winter. 

The  larvae  (fig.  125,  e),  are  footless  maggots,  whitish  in 
colour,  and  have  thirteen  segments,  of  which  the  first  is 
provided  with  a  buccal  apparatus  and  antennae.  The 
buccal  apparatus  is  formed  for  masticating.  They  are 
always  found  in  dry  places,  dust,  sand,  or  clothing.  At 
the  end  of  about  ten  days  the  larva  ceases  to  feed  and 
becomes  immobile  and  forms  about  itself  a  small  cocoon, 
to   which  dirt,  sawdust,    &C.    adhere.       In    about    eleven 


DISSECTION   OF    FLEAS  287 

days  the  larva  is  transformed  into  a  nymph,  which  has 
three  pairs  of  legs  and  resembles  the  perfect  insect. 
These  nymphs  after  a  further  period  of  about  twelve 
days  are  transformed  into  perfect  insects  and  come  out 
of  the  cocoon. 

The  whole  process  is  accomplished  in  about  a  month 
in  summer  and  six  weeks  in  winter. 

Dissection. — This  is  best  carried  out  in  normal  saline 
solution,  using  needles  with  very  fine  points.  With  a 
simple  lens  the  dissection  is  very  difficult — a  stereoscopic 
microscope  is  almost  an  essential. 

The  flea  is  transfixed  through  the  head  and  held  by  the 
left-hand  needle.  The  point  of  the  right-hand  needle  is 
then  inserted  under  the  edge  of  the  third  or  fourth  abdo- 
minal segment  and  the  chitinous  covering  peeled  off. 
The  internal  organs  float  out  in  the  salt  solution  and  may 
be  further  separated. 

Internal  Anatomy. — The  mouth  is  situated  at  the  base  of 
the  bulb  of  the  ventral  portion  of  the  epipharynx  and 
forms  the  commencement  of  the  alimentary  canal. 

The  pharynx  extends  from  the  mouth  to  the  oesopha- 
geal commissure.     It  is  aspiratory  in  function. 

There  are  two  salivary  glands,  each  consisting  of  two 
lobes,  and  they  lie  on  each  side  of  the  anterior  end  of  the 
stomach  embedded  in  the  fat  body. 

At  the  junction  of  the  pharynx  and  the  stomach  is  an 
organ  to  which  the  name  of  "  gizzard"  has  been  applied, 
It  is  suggested  that  its  function  is  to  prevent  regurgitation 
of  fluids  from  the  stomach. 

The  stomach  consists  of  a  basement  membrane,  two 
oblique  layers  of  muscle  fibre  and  a  lining  layer  of  cubical 
cells. 

Classification. — Fleas  are  divided  into  three  families, 
Pulicidce,  Sarcopsyllidce,  and  Vermipsyllidce,  of  which  onlv 
the  first  two  are  of  interest  to  us. 

Family  PuLlClDiE. — Generally  speaking  larger  than 
unimpregnated  Sarcopsyllidce.  Head  relatively  small, 
round   above   and   frequently   armed   with  combs  upon 


3=  t".  3=1: 


r-^m 


Fig.  125. — a,  Sarcopsylla  penetrans  ; 
d,  Ceralophyllus  ; 


/>,  I  u lex  irritans  ;        r,  Cienocephalus  trraticep: 
e,  Larva  of  Pulex ;        f,  Pulex  cheopis. 


CLASSIFICATION  289 

the  side,  or  along  its  inferior  border.  Labial  palps 
always  consisting  of  four  segments.  Thorax  longer  than 
in  Sarcopsyllidce,  in  some  genera  having  a  comb  of  spines 
at  the  posterior  border  of  the  prothorax,  sometimes  also 
at  that  of  the  metathorax.  In  others  there  are  combs  at 
the  posterior  border  on  one  or  more  of  the  segments  of 
the  abdomen.  The  females  never  become  fixed  in  the 
tissues  of  the  host,  nor  does  the  abdomen  become  greatly- 
distended  as  in  Sarcopsyllidce. 
Genera  of  family  Pulicidce  : — 

A.  Eyes  well  developed. 

Comb  along  inferior  border  of  head  and  another 
along  posterior  border  of  prothorax  Ctenocephalus. 

Comb  along  posterior  border  of  prothorax.  No 
comb  on  head.  Third  joint  of  antenna  com- 
pletely segmented CcratopJiyllus. 

No  combs.  Third  joint  of  antenna  segmented 
dorsally  only  Pulex. 

B.  Eyes  rudimentary  or  absent. 

Comb  on  inferior  border  of  head  and  another 

along  posterior  border  of  prothorax.     Spines 

on  posterior  aspects  of  tibiae  arranged  in  pairs     CtenopJithahnus. 
Comb  on  inferior  border  of  head  and  another 

along  posterior  border  of  prothorax.     Spines 

on    posterior  aspects    of  tibiae  arranged  in  a 

close  set  row  Ctenopsylla. 

Comb  on  inferior  border  of  head  and  another 

along  posterior  border  of  prothorax.     Combs 

also   on  one   or    more   abdominal   segments. 

Body  covered  with  hairs  and  small  spines  ...     Hystrichopsylla. 

Pulcx  cheopis  \ — This  flea  has  been  described  under 
various  names,  P.  pallidus,  P.  murinus,  &c.  It  is  the 
commonest  flea  found  on  rats  in  warmer  climates,  and 
in  some  localities  is  the  only  flea.  It  has  been  found  in 
Marseilles,  Italy,  Sydney,  Philippines,  India,  Sudan,  South 
Africa  and  South  America. 

It  is  a  non-pectinated  flea,  i.e.  without  combs,   resem- 

1  This  flea  has  recently  been  removed  from  the  genus  Pulex  and 
placed  in  the  genus  Xe?iopsylla.  The  latter  genus  is  distinguished 
from  the  former  by  the  character  of  the  lines  on  the  meso-sternum. 

19 


290  SARCOPSYLLIDjE 

bling  the  human  flea,  hut  differs  from  it,  among  other 
characteristics,    in    the    body   being    lighter   in    colour, 

yellowish  instead  of  brownish,  and  in  the  position  of  the 
ocular  hair,  which  is  situated  in  front  of  and  above  the 
eye,  while  in  P.  irritans  it  is  situated  in  front  of  and  below 
the  eye. 

Other  rat-fleas  are  Ceratophyllus  fasciattis,  Ctenopsylla 
musculi,  Ctenocephalus  serraticeps,  Hyslrichopsylla  talpce, 
as  well  as  many  less  common  species. 

Family  Sakcopsyllid.e. — Body  small,  head  relatively 
large,  angular  or  rounded  above,  never  armed  with  spine-. 
Labial  palps  unsegmented.  Thoracic  segments  small. 
Abdomen  variable,  more  or  less  swollen  in  the  fertilised 
female.  Never  any  combs  on  thorax  or  abdomen.  The 
fertilized  female  burrows  into  the  skin  of  the  host. 

This  family  comprises  two  genera,  Sarcopsylhi  and 
Rhynchopsylla. 

The  genus  Sarcopsylla  contains  the  important  parasite, 
Sarcopsylla  penetrans — the  Jigger  or  Chigoe.  It  is  a  small 
flea,  the  male  measuring  about  1  mm.  in  length,  and  the 
unimpregnated  female  about  the  same  size.  The  general 
colour  of  the  body  is  brownish.  The  head  relatively  to 
the  body  is  large  ;  its  upper  surface  slopes  obliquely  in 
front,  and  joins  the  lower  surface  at  an  acute  angle.  The 
eye  is  situated  in  the  front  part  of  the  head  at  the  border 
of  the  fossa  of  the  antenna. 

The  perforating  and  suctorial  mouth-parts  are  well 
developed,  but  the  maxillae  are  very  small  quadrilateral 
structures,  only  seen  with  difficulty. 

The  three  thoracic  segments  are  very  small. 

The  males  and  unimpregnated  females  are  parasitic  on 
man  occasionally,  for  the  purpose  of  sucking  blood. 
The  impregnated  female,  however,  bores  her  way  into 
the  skin,  particularly  about  the  feet,  and  the  abdomen  of 
the  insect  undergoes  great  distension  following  the 
development  of  the  eggs,  the  head  and  thorax  remaining 
unchanged. 

The  eggs  develop  on  the  soil,  and  the  metamorphosis 
is  similar  to  that  which  obtains  in  other  fleas. 


ANOPLEURA  291 

The  Jigger  is  widely  distributed  in  the  Tropics.  Origin- 
ally found  only  in  Central  and  South  America,  it  was 
introduced  into  West  Africa  about  1872,  and  in  a  few 
years  had  become  disseminated  throughout  the  greater 
part  of  Africa. 

Another  member  of  this  genus,  S.  gallinacea,  attacks 
fowls,  particularly  about  the  head,  and  causes  great 
destruction  among  them.  S.  gallinacea  does  not  bury 
itself  in  the  skin  so  completely  as  does  S.  penetrans,  nor 
does  the  abdomen  become  so  greatly  distended,  as  the 
eggs  are  laid  one  by  one  as  they  mature. 

Order  ANOPLEURA  or  SlPHUNCULATA. 

These  must  be  distinguished  from  Melophaga  and  bird 
lice.  The  former  belong  to  the  order  Diptera,  whilst  the 
latter  are  degenerate  N  enroptcra  and  do  not  suck  blood 
but  live  on  epidermis. 

The  members  of  the  order  Anoplenra  suck  blood  by 
means  of  a  sort  of  double  tubular  proboscis,  armed  with 
spines  which  are  usually  retracted  under  the  head.  Eyes 
simple.  Antennae  of  3  to  5  joints.  Thorax  composed 
of  three  segments.  Three  pairs  of  legs  terminated  by 
strong  claws. 

There  are  six  to  nine  segments  in  the  abdomen  ter- 
minated by  two  basal  lobes  in  the  female  and  a  large 
triangular  penis  in  the  male. 

Family  Pediculid.-e. — The  body  is  flattened.  The 
rostrum  is  provided  with  barbed  hooklets.  The  antennae 
are  inserted  in  a  kind  of  sinus  in  the  front  of  the  head, 
and  are  composed  of  three,  four,  or  five  segments,  the 
number  of  these  being  used  for  the  separation  of  genera. 
The  three  thoracic  segments  are  distinctly  separated. 
The  abdomen  comprises  six  to  nine  segments.  Legs  are 
terminated  by  one  or  two  long  claws. 

The  eggs  are  pyriform  in  shape,  fixed  by  their  smaller 
end  to  the  bases  of  the  hairs  of  the  host;  the  larger  end 
is  provided  with  an  operculum  which  the  young  split  off 


292 


PEDICUL] 


in  emerging.  After  the  eggs  arc  laid  the  young  are 
hatched  out.  They  resemble  the  adults  and  mature 
rapidly.     There  is  no  metamorphosis. 

The    family    comprises    several     genera,    of    which    we 
consider  only  Pediculus  and  Phthirius. 

(Distinct   neck  ...     Pediadus. 
'Thorax   narrower 
Antennre  with  five  seg-  !      than  abdomen      I  No  distinct  neck     Hcrmatopiuus. 
ments.     Legs  with  a  ] 
single  claw  (Thorax  broader  than  abdomen         ...     Phthirius. 


Fig.  126.  —Pediculia  veslimenti. 


Pediculus  capitis  (Head-louse). — The  male  is  1  to  2 
mm.  in  length,  the  female  somewhat  larger.  The  head 
is'  triangular  in  shape,  the  thorax  narrower  than  the 
abdomen.  The  abdomen  has  eight  segments,  is  pig- 
mented all  round  and  is  hairy. 

The  female  lays  fifty  to  sixty  eggs  which  hatch  out  in 
about  seven  days.  The  young  are  able  to  reproduce  in 
seventeen  to  twenty  days  after  birth. 

Pediculus  veslimenti. — (Body-louse). — Male  2  to  3  mm. 
in    length,    females    larger.      Head    somewhat    rounded. 


PKDICULI 


293 


Antennae  longer  than  in  P.  capitis.  The  thorax  is  as  wide 
as  the  abdomen,  which  is  not  hairy  or  pigmented. 

The  female  lays  seventy  to  eighty  eggs,  which  hatch 
out  in  three  to  eight  clays. 

Phthirius  inguinalis  (Crab-louse). — The  genus  Phthirius 
includes  a  single  species.  This  species  is  peculiar  to  man. 
It  may  be  found  in  any  hairy  part  except  the  scalp,  but  is 
most  commonly  found  on  the  pubic  hairs. 

Male  o-8  to  ro  mm.  in  length  :  female  1  to  1*5  mm. 
The  head  is  large  and  provided  with  two  long  antennae. 
Thorax  is  broader  than  the  abdomen  and  shows  no  trace 
of  separation  from  it.  There  are  six  segments  in  the 
abdomen. 


Fig.  127. — Phthirius  inguinalis. 

The  female  lays  ten  to  fifteen  eggs,  which  hatch  out  in 
six  to  seven  days. 

An  interesting  observation  made  by  Murray,  that  the 
pediculi  of  different  races  differed  in  colour  and  even  in 
certain  anatomical  details,  has  since  been  confirmed  by 
many  observers.  This  is  regarded  as  a  kind  of  protective 
mimicry.  From  observations  on  these  parasites  in  the 
Federated  Malay  States,  we  believe  that  the  pediculi  of 
one  race,  for  example  the  Chinese,  do  not  pass  readily 
if  at  all,  to  another  race,  for  example,  the  Tamils. 

Order  Hemiptera  or  Rhyncota. 
This  order  includes  insects  of  very   dissimilar  charac- 
teristics.    The  rostrum  is  the  feature  by  which  they  are 


:  >4 


BED-B1  GS 


most  easily  distinguished.     This  organ  is  a  modification 

of  the  interior  lip,  it  is  tubular  in  character,  and  in  a 
state  <>!'  repose  is  folded  up  under  the  head  and  thorax. 
The  rostrum  encloses  the  hair-like  penetrating  parts. 

There  are  two  important  families  belonging  to  this 
order— Cimicida,  Reduviidce. 

Family  Cimicid.k. — This  family  of  the  Hemiptera  be- 
longs to  the  division  known  as  Heteropteta,  as  there  is 
a  marked  difference  between  the  two  pairs  of  wings. 

The  family  is  characterized  by  the  absence  of  ocelli, 
by  the  wings  being  rudimentary,  very  short  and  broad,  so 
that  the  abdomen  is  left  uncovered.  The  head  is  short 
and  broad,  and  the  rostrum  is  received  in  a  groove 
beneath  the  head.  Tarsi  are  three-jointed.  Antennae 
long  and  composed  of  four  joints. 


Fig.  I2§. — Cimex  lectuarius. 

Genus  Cimex  comprises  at  least  two  species  parasitic 
on  man,  C.  lectuarius  and  C.  rotundatus.  Considerable 
interest  centres  about  these,  in  view  of  Patton's  suggestion 
that  they  are  the  carriers  of  the  parasites  of  kala-a/ar, 
and  the  older  hypotheses  that  they  may  convey  relapsing 
fever  and  leprosy.  There  is  no  proof  in  support  of  these 
hypotheses. 

Cimex  lectuarius. — This  insect  is  oval  in  form, brownish- 
red  in  colour,  with  a  much  flattened  body.  It  measures 
4  to  5  mm.  in  length  and  3  mm.  in  breadth.  Eight 
abdominal  segments. 


BED-BUGS  295 

The  eves  are  simple  and  there  are  no  ocelli.  The 
elytra  are  rudimentary  and  lie  on  the  metathorax.  The 
prothorax  is  semilunar  in  shape,  its  anterior  angles  being 
considerably  developed  and  coming  close  up  to  the  eyes. 

The  female  lays  about  fifty  whitish  eggs,  three  or  four 
times  a  year.  The  complete  development  takes  about 
eleven  months,  during  which  time  it  casts  its  skin  five 
times.     The  young  larvae  are  at  first  pale  white. 

Bed-bugs  live  in  cracks  in  the  walls,  under  carpets, 
behind  pictures,  wall  papers,  &c,  and  only  come  out  at 
night  to  suck  blood,  which  is  their  only  food.  They  may 
live  from  five  to  ten  weeks,  or  even  longer,  without  food. 
The  average  duration  of  life  is  three  to  four  months. 

Cimex  rotundatus. — This  species  was  originally  described 
from  the  island  of  Reunion  in  1852.  Patton  has  shown 
that  it  is  distributed  throughout  India,  Burmah  and  Assam. 

It  is  rather  smaller  than  C.  lectuarius,  and  in  general 
outline  less  rounded.  The  main  feature,  however,  is  the 
character  of  the  prothorax,  which  has  well-rounded 
borders  and  gives  to  the  animal  an  appearance  of 
rotundity.  The  anterior  angles  of  the  prothorax  do  not 
come  up  so  close  to  the  eyes  as  in  C.  lectuarius. 

Other  species  of  Ciuicx  described  are  C.  ciliatus  from 
Kasan,  Russia,  which  is  smaller  than  the  common  bed- 
bug, yellowish-red  in  colour,  and  thickly  covered  with 
hairs,  C.  columbarius,  C.  himndinus,  and  C.  inodorus, 
which  have  been  found  attacking  birds. 

Family  Reduviid^e. — This  family  is  a  large  one  and 
of  world-wide  distribution.  The  members  of  this  family 
are  mostly  of  large  size  and  are  distinguished  from  the 
cimicidae  by  the  fact  that  the  rostrum  curves  backwards 
under  the  head  and  does  not  lie  closely  applied  to  the 
under  surface. 

Some  of  the  Reduviidae  are  useful  as  they  are  predatory 
on  other  insects. 

To  the  important  genus  Conorrhinus  belongs  C. 
sanguisuga,  a  large  South  American  bug.  This  sucks 
blood  and  has  been  shown  to  be  the  host  of  a  human 
trypanosome,  T.  cruzi. 


></> 


CHAPTER  XVI. 

Arachnoidea — Ticks,  Mites,  Porocephalus. 
Crustacea — Cyclops. 

The  Arachnoidea  are  a  class  of  the  Arlhropoda  readily 

distinguished  from  insects,  as  they  have  four  pairs  of  legs 
in  the  adult  stage.  The  head  and  thorax  are  usually  fused 
into  a  compact  mass,  the  cephalothorax.  The  abdomen  is 
generally  without  appendages  ;  it  may  be  segmented  or 
unsegmented  ;  it  is  generally  distinct  from,  but  may  be 
fused  to  the  cephalothorax.  Compound  eyes  such  as 
occur  in  insects  are  never  present — the  eyes  when  present 
are  always  simple.  Respiration  may  be  by  means  of 
tracheae  or  cutaneous.  The  genital  orifice'is  in  the  middle 
or  in  the  anterior  half  of  the  ventral  surface  and  is  dis- 
tinct from  the  alimentary  canal.  The  sexes  are  distinct. 
In  most  cases  the  newly  hatched  young  arc  essentially 
like  the  adults. 

The  class  Arachnoidea  is  divided  into  a  number  of 
orders,  including  Scorpionidai  (scorpions),  Psendoscorpion- 
iihu  (book  scorpions),  Pedipalpi  (whip-scorpions),  Ara- 
iicidcu  (spiders),  Acarina  (mites  and  ticks),  and  certain 
aberrant  orders,  as  Linguahdida  or  Pentastomiidai. 

Order  ACARINA  (Mites  and  Ticks). — These  are  small 
Arachnoids,  usually  parasitic.  They  occur  in  earth,  in 
fresh  or  salt  water,  or  on  animals  or  plants.  They  feed 
on  the  organisms  they  infest  or  upon  organic  matter. 

The  abdomen  and  cephalothorax  form  a  fused  mass. 
The  mouth-parts  are  formed  for  biting  or  for  puncturing 
and  sucking,  according  to  the  mode  of  life.  Respiration 
maybe  cutaneous,  but  in  most  there  are  tracheae  with  two 


TICKS 


297 


stigmata.     Many  of  the  young  have  only  three  pairs  of 
legs  when  hatched,  but  alter  moulting  have  four  pairs. 

The  Acarina  which  cause  disease  or  act  as  carriers  of 
disease  are  divided  into  families  as  follows  : — 


Non-vermi- 
orm  acarina 


/(a)  Legs  inserted  Legswiih  Tracheae  open- 
directly  into  in-  six  joints  ing  in  the 
tegument  posterior  part 

of  body 


(6)  Legs    articulated 
on  distinct  epimerae 


Vermiform    Legs    articulated    Legs  with    No  trachea 
acarina  on  distinct  epi-       three 

merae  joints 


'Chelicerce  with    IxoJida. 
hooklets 

Chelicem?      di-    Gaiuasiihe. 
■  dactylous    or 
styliform 


/'Tracheae  open- 
ing in  ante- 
rior part  of 
body 

,No  trachea? 


Trombididce. 


Sarcoptidce. 


Cheliceras  styli- 
form or  with 
hooklets  ; 
palps  free 

Chelicerce  di- 
dactylous ; 
palps  cylin- 
drical and 
adherent  to 
inferior  lip 


Chelicerce  styli-    Demodicidtz. 
form  ;    palps 
with  hooklets 


IXODID^E. 

The  family  Ixodidce  is  composed  of  comparatively  large 
Acarines  with  leathery  skins.  They  have  flattened  bodies, 
but  after  sucking  blood  become  much  distended.  The 
mouth-parts,  which  are  characteristic,  comprise  a  median 
penetrating  organ,  the  hypostome,  armed  with  recurved 
teeth,  two  rod-like  organs  surrounded  at  their  bases 
by  a  sheath,  the  chelicerce,  each  of  which  is  terminated  by 
a  process  bearing  large  hooked  teeth.  On  either  side  of 
the  chelicene  and  hypostome  lies  a  four-jointed  palp 
which  in  some  genera  is  closely  applied  to  the  piercing 
organs  and  appears  to  act  as  a  sheath  for  them,  but  in 
other  genera  is  quite  free  (fig.  132). 

Each  leg  consists  of  six;  main  segments  known  respec- 
tively as  coxa,  trochanter,  femur,  patella,  tibia  and  tarsus. 
The  trochanter  may  have  spines  or  teeth.  The  tarsus 
bears  on  its  terminal  segment  two  claws  (iig.  129).  There 
are  two  sub-families,  Ixodince  and  Argasince. 


298 


TICKS 


In  the  sub-family  Ixodince  the  skin  of  the  dorsal  surface 
is  thickened  to  form  a  hard,  leathery,  chitinous  plate,  the 
scutum  or  dorsal  shield,  which  in  the  male  covers  almost 
the  whole  of  the  dorsum,  while  in  the  female  it  covers 
only  the  anterior  portion  of  the  dorsum.  This  arrange- 
ment serves  to  distinguish  males  from  females.  In  the 
sub-family  Argasince  these  shields  are  not  present. 


Fig.  129. 


Eyes^are  not  always  present.  They  consist  of  a  simple 
lens  onlv  and  may  he  globular  or  flat.  In  the  Ixodince 
they  are  situated  at  the  margins  of  the  dorsal  shield;  in 
the  Argaslnce  on  a  ridge  above  the  coxa?. 

The  stigmata  or  openings  of  the  tracheal  system  are 
situated    behind    the   level   of   the    fourth   pair   of    legs   in 


Fig.  130.—  Ixodina  (Female),     a,  Dorsal  aspect,  showing  shield;    b,  ventral  aspect. 


300  EXAMINATION    OF  TICKS 

Ixodince  and  between  the  third  and  fourth  pair  of  legs  in 
the  Argasince. 

The  genital  orifice  is  situated  in  the  middle  line  on  the 
ventral  surface,  a  short  distance  behind  the  rostrum  at 
the  level  of  the  second  and  third  coxae. 

In  the  middle  line,  behind  the  level  of  the  last  pair  of 
legs,  is  the  valvular  anus.  On  either  side  of  the  anus  in 
the  males  of  certain  genera  are  situated  thickenings  of 
the  cuticle  known  as  ddanal  plates  :  the  number,  shape, 
and  position  of  these  are  of  value  in  classification. 

Examination  of  Ticks. — Ticks  can  be  examined  living 
or  dry,  and  the  main  points  clearly  made  out  with  the 
aid  of  a  hand  lens.  The  dorsal  and  ventral  aspects  must 
be  examined  in  turn.  The  points  to  observe  on  the 
ventral  surface  are  along  the  median  line,  the  rostrum, 
with  the  palps  on  each  side;  the  opening  of  the  genital 
organs  in  the  anterior  half  of  the  body  and  the  anal 
opening  in  the  posterior  half,  and  any  deep  furrow  in 
front  or  behind  this  anus,  anal  furrow.  At  the  sides  the 
position  and  any  special  markings  on  the  coxae  should 
be  noted  ;  the  respiratory  area,  stigma,  anterior  or  pos- 
terior to  the  fourth  coxae  and  its  shape,  and  the  presence 
or  absence  of  any  "  shields  "  or  chitinous  plates  on  each 
side  of  the  anus,  ddanal  shields  or  plates. 

On  the  dorsal  aspect  the  presence  or  absence  of  a  dorsal 
shield  or  scutum  should  be  noticed,  and  its  extent  ;  as  to 
whether  it  covers  the  whole  of  the  back  or  the  anterior 
part  only.  Any  markings,  bosses  or  protuberances,  and 
how  far  these  form  a  continuous  pattern  to  the  edge  of 
the  dorsum,  or  vary  at  the  margins.  Eyes  should  be 
looked  for. 

The  details  in  the  structure  of  the  mouth-parts  are  best 
seen  in  specimens  rendered  translucent  by  treatment  with 
alkalies.  In  smaller  ticks,  if  not  distended  with  blood  or 
eggs,  boiling  for  a  few  minutes  in  a  10  per  cent,  solution 
of  caustic  soda  will  suffice,  but  it  is  better  to  leave  for  a 
longer  time  in  the  solution.  Many  ticks  contain  chro- 
matin and    are  not  readily  decolorised.     After  the  treat- 


Fig.   \i\.  —  Txodina  (Males),     a,  Dorsal  aspect,  showing  dorsal  shield  ;  b,  ventral  aspect 
in  species  wiih  adanal  plates  ;  c,  ventral  aspect  in  species  without  adanal  plates. 


302  ANATOMY   OF  TICKS 

raent  with  caustic  soda  they  should  be  well  washed,  and 
mav  be  examined  mounted  in  glycerine  or  dehydrated 
with  alcohol  and  oil  of  cloves  and  mounted  in  Canada 
balsam. 

Dissection  of  Ticks. — Christophers  recommends  the  fol- 
lowing method  :  Holding  the  tick  between  linger  and 
thumb,  with  a  pair  of  sharp  scissors  snip  fine  slices  from 
the  edges.  Gently  wash  in  normal  saline  solution  and 
place  in  a  small  dish  containing  the  same  solution.  By 
using  one  pair  of  forceps  to  seize  the  ventral  flap  and 
another  the  dorsal  flap  at  their  posterior  ends,  the  latter 
may  be  dragged  forward  over  the  head,  leaving  the 
viscera  attached  in  situ  to  the  ventral  surface.  The  chief 
anatomical  features  can  be  made  out  by  displacing  the 
larger  organs  and  removing  with  fine  forceps  the  tissues 
which  hold  these  in  position. 

Internal  Anatomy  of  Ticks. — The  chitinous  pharyngeal 
pump,  which  opens  into  the  mouth,  leads  posteriorly  to 
a  narrow,  straight  oesophagus.  The  oesophagus,  after 
perforating  the  central  nerve  ganglion,  enters  the  enor- 
mous saccular  portion  of  the  alimentary  canal  which, 
with  its  diverticula,  forms  the  great  bulk  of  the  body 
contents.  Posteriorly  an  extremely  fine  canal  joins  the 
central  saccular  gut  with  the  rectum.  Opening  into  the 
rectum  on  either  side  is  a  Malpighian  tubule.  These 
latter  are  two  fine  white  or  transparent  tubules  of  great 
length  which,  after  a  complicated  course  among  the 
viscera,  end  blindly  in  the  anterior  part  of  the  body. 
The  rectum  has  several  diverticula  and  terminates  in 
the  anus. 

The  salivary  glands,  two  in  number,  lie  over  the  bases 
of  the  first  two  pairs  of  legs.  There  is  a  central  duct 
which  arises  near  the  free  extremity,  and  passing  through 
the  whole  length  of  the  gland  becomes  the  salivary  duct. 
The  salivary  ducts  open  into  the  mouth.  In  the  female 
the  single  ovary  lies  upon  the  diverticula  of  the  alimen- 
tary canal  in  the  posterior  portion  of  the  body.  Leading 
from  it  on  either  side  are  the  long  coiled  oviducts.     In 


LIFE-HISTORY   OF   TICKS  303 

front  of  the  ovary  is  the  spermatheca.  In  the  male,  in  the 
same  position  as  the  ovary  in  the  female,  is  a  thin 
delicate  tubule,  the  testis. 

Life-history. — The  ova  of  ticks  are  laid  in  recesses  in 
the  soil ;  in  the  Ixodince  the  number  of  these  is  2,000  to 
4,000,  in  the  Argasince  a  few  hundreds  only.  After  a 
variable  period  the  six-legged  larva  is  hatched  out.  The 
larvae  of  the  Ixodince  attach  themselves  at  the  first  oppor- 
tunity to  some  vertebrate,  from  which  they  suck  blood. 
In  some  ticks  the  larva,  having  gorged  itself  with  blood, 
drops  off  upon  the  ground,  and  after  a  few  days  casts  its 
skin  and  emerges  as  the  eight-legged  nymph,  which  differs 
from  the  adult  form  mainly  in  the  absence  of  a  genital 
opening.  The  nymph  in  turn  attacks  a  fresh  host,  and, 
having  fed,  drops  off,  and  after  moulting  becomes  the 
adult  tick.  H cemaphysalis  leachi  undergoes  its  develop- 
ment in  this  way.  In  other  ticks,  such  as  Boophilus 
annulatus,  the  metamorphosis  from  larva  to  nymph  takes 
place  on  the  host.  The  nymph  may  or  may  not  leave  the 
host  before  reaching  the  adult  stage.  In  Ornithodoros 
moubata  and  0.  savignyi  the  larvae  do  not  suck  blood. 

Ticks  as  Carriers  of  Disease. — Ticks  are  known  to  be 
carriers  of  several  species  of  hasmatozoal  parasites  be- 
longing to  the  genera  Piroplasma  and  Spirochceta.  In 
man  a  spirochaete  (S.  duttoni)  which  is  the  causative 
agent  in  African  relapsing  fever  is  carried  by  a  tick, 
Ornithodoros  moubata,  and  an  analogous  disease  of  fowls, 
also  due  to  a  spirochaete,  is  transmitted  by  Argas 
persicus.  In  mammals  a  number  of  diseases  conveni- 
ently grouped  under  the  name  Piroplasmoses  are  carried 
by  ticks  belonging  to  the  sub-family  Ixodince. 

It  is  important  to  note  the  stage  in  its  development  at 
which  a  tick  may  transmit  parasites,  as  this  differs  accord- 
ing to  the  species  of  parasite  and  the  species  of  tick  con- 
cerned. For  example,  larvae  of  Boophilus  annulatus, 
from  an  infected  mother,  are  able  to  transmit  Texas  fever, 
while  in  the  case  of  Ha'inaphysalis  leachi,  one  of  the 
carriers  of  canine  piroplasmosis,  the  larvae  and  nymphs 


,04 


CLASSIFICATION    OK   TICKS 


are  not  infective,  and  only  the  adult  can  convey  the  dis- 
ease ;  again,  in  the  case  of  Amblyomma  hebrceum,  which 
convevs  the  parasite  of  heartwater  in  sheep  and  goats, 
the  nymph  is  infective  if  fed  as  a  larva,  and  the  adult  if 
fed  as  a  nymph. 

Systematic  Classification  of  Ticks. 

Rostrum  on  ventral  surface  of  body  not  visible 
from  above  in  the  adults.  No  dorsal  shields 
or  adanal  plates.  Stigma  in  front  of  fourth 
coxa.     Palps  free  or  not  grooved Sub-family  Argasiiice. 


Fig.  132. — Mouth-parts  of  Ornithodoros. 

Rostrum  terminal.  Dorsal  shields  present. 
Adanal  plates  in  males  of  some  genera  only. 
Stigma  posterior  to  fourth  coxa.  Palps 
grooved  on  internal  aspect  and  usually  closely 
applied  to  rostrum    Sub-family  Ixodimr. 

Sub-family  Argasinee. 

Body  with  sharp  edges.  No  eyes.  No  deep 
furrows  on  ventral  surface.  Skin  wrinkled. 
Pattern  of  marking  on  the  edge  differs  from 
that  on  the  remainder  of  the  dorsum  ...Genus  Argas. 

Body  with  thick  edges.  Eyes  present  in  some 
species.  Often  deep  furrows  on  ventral  sur- 
face. Skin  covered  with  bosses  and  the 
pattern  of  these  is  continued  to  the  edge.. .Genus  Ornithodoros. 


IXODIX.E 


3°5 


Sub-family  Ixodince. 
Palps  long,   the  second  joint  much  longer   than 

broad    Group  I.  Ixodce. 

Palps  short,  the  second  joint  much  broader  than 

long Group  II.  Rhipicephalcc. 


Fig.  133. — Mouth-parts  of  Ixodes. 


Fig.  134. — Mouth-parts  of  Rhipicephalus. 

Group    I.     Ixodince. 

1.  Anal  furrow  in  front  of  anus.     No  eyes  ...Genus  Ixodes. 

2.  Anal    furrow   behind    anus,    often    continued 
laterally  to  genital  furrow  (fig.  130). 

(a)  Adanal  plates  present  in  $.    Eyes  present 

Genus  Hyalotnma. 
20 


306  IXODIX.K — ORXITHODOROS 

(l>)  No  anal  plates  in  3' 

(a)   With  eyes C.enus  Amblyomma. 

ifi)  Without  eyes  Genus  Aponovwui. 

Group  II.     Rhipicephalce. 
.1.  No  eyes  present. 

No  adanal  plates  in  J.     Second  segment  of 
palp  has  a  well-marked  lateral  projection... 

Genus  Hcemaphysalis. 
i.  Eyes  present. 

(a)  No  adanal  plates  in  $  .  Base  of  rostrum 
quadrilateral.  Coxae  of  last  pair  of  legs 
are  always  large Dermatocentor. 

(/>)  With  adanal  plates  in  <?•     Base  of  rostrum 

hexagonal  with  well-marked  lateral  angle. 

(i)  Two  adanal  shields.    Pre-anal  furrow 

present.      Stigmata,  comma-shaped     Rhipiccplialus. 

(2)  Similar  to  Rhipicephalus  except  for 
stigmata  which  are  small  and  circular     Boophilus. 

(3)  Similar   to    Boophilus   except    that 
joints  of  legs  and  especially  of  the 

hind  legs  are  of  enormous  breadth     Margaropus. 
(c)  No  adanal  plates.      Coxns  of  last  pair  of 
legs  produced  into  enormous  spines:  more 
marked  in  $  than  in  5     Rhipicentor. 

Omithodoros  moubata. — Length  about  8  mm.,  width 
6  to  7  mm.  General  shape  of  the  body  ovoid,  somewhat 
wider  behind  than  in  front ;  yellowish-brown  in  colour 
when  young,  greenish-brown  when  adult.  Integument 
studded  with  small  tubercles.  On  the  dorsal  surface 
three  pairs  of  grooves  running  obliquely  downwards  and 
inwards  towards  posterior  end.  Above  the  bases  of  the 
legs  a  longitudinal  supra-coxal  groove.  On  the  ventral 
surface  there  is  a  deep  pre-anal  groove  joining  the  supra- 
coxal  grooves  laterally;  behind  the  anus  three  pairs  of 
longitudinal  grooves. 

There  are  no  eyes  present.  Stigma  semilunar  in  front 
above  the  supra-coxal  groove.  Fourth  leg  one  and  a 
half  times  as  long  as  the  first;  tibiae  and  tarsi  of  first 
three  pairs  with  three  teeth  on  the  upper  side. 

This  species  is  widely  distributed  in  Central  Africa  from 


Fig.   135.—  Ornithodoros  savignyi.     a,  Ventral  aspect  ;  b,  dorsal  aspect  ;  r,  lateral 
aspect  between  second  and  third  pair  of  legs. 


3o8 


DEMODEX 


cast  to  west.  Another  species,  0.  savignyi,  in  which  eyes 
are  present,  lias  been  found  in  Somaliland,  German  Easl 
Africa,  the  Congo  and  India.  (Fig.  135,  0.  savignyi, 
dorsal  and  ventral  surface.) 

Family  Demodicidce. — Body  small,  vermiform,  com- 
prising an  anterior  part  provided  with  legs  and  a 
posterior  part  showing  transverse  striation.  Mandibles 
styliform,  palps  consisting  of  three  segments,  the  last  of 
which  has  a  curved  hooklet.  No  eves.  Four  pairs  of 
legs.  No  stigmata.  Larvae  have  three  pairs  of  rudi- 
mentary legs  or  may  be  footless. 


Fig.  1 36. — Demodex  folliculantm. 


These  animals  are  parasites  of  the  hair  follicles  and 
sebaceous  glands  of  the  skin  of  mammals. 

Demodex  follicularum  (fig.  136)  is  a  common  parasite 
in  man,  and  may  cause  inflammation  by  obstructing 
excretory  gland  ducts. 

The  male  is  about  3  mm.  in  length  and  the  female 
4  mm.  in  length. 


Order    LlNGUATULlD.E. — Probablv 


degenerate    Arach- 


LINGUATULID/E  309 

uoidea.  They  are  worm-like  in  form  and  externally 
annulated  ;  there  is  no  distinction  between  head,  thorax, 
and  abdomen.  No  oral  appendages  are  present,  but  there 
are  two  pairs  of  movable  hooks  near  to  the  mouth,  which 
are  regarded  as  remains  of  the  antennae  and  palpi. 
Alimentary  canal  simple,  no  Malpighian  tubes.  There 
are  no  sense  organs  or  tracheae. 

The  sexes  are  distinct ;  the  males  smaller  than  the 
females. 

Adults  live  in  the  nasal  cavity,  frontal  sinus,  or  lungs 
of  the  dog,  wolf,  and  other  animals;  occasionally  they 
are  found  in  man. 

The  females  lay  eggs,  which  if  taken  up  by  an  inter- 
mediate host  give  rise  to  embryos.  These  embryos  pass 
from  the  intestine  to  the  liver  or  lung  where  they  encyst, 
moult,  and  pass  through  a  larval  stage,  in  which  the 
characters  of  the  adult  are  developed.  Finally  they  reach 
the  nasal  cavities  of  the  same  or  a  new  host  and  become 
sexually  mature. 

Porocephalus  arm-Mains.  The  larval  form  originally 
known  as  Pentastomum  constrictum  is  probably  a  Poro- 
cephalus. It  has  been  found  on  several  occasions  en- 
cysted in  the  livers  of  negroes  in  Africa.  The  number 
of  rings  is  never  more  than  twenty-two,  which  corresponds 
to  the  number  of  rings  in  the  larval  forms  of  Porocephalus 
armillatus.  According  to  Sambon  the  adult  forms  of 
this  group  occur  in  the  lungs  of  the  Royal  Python  and 
in  those  of  the  Nose-horned  Viper. 

Class  Crustacea  are  not  as  a  rule  carriers  of  disease. 
The  larger  ones  are  edible,  and  occasionally  some, 
especially  prawns  and  shrimps,  are  believed  to  harbour 
typhoid  bacilli.  From  the  point  of  view  of  carriers 
of  disease  the  Copepoda  are  of  most  importance. 
Order  COPEPODA  :  Elongated  crustaceans,  usually  with 
distinct  segments.  No  dorsal  shell.  Five  pairs  of 
biramose  thoracic  appendages,  the  last  of  which  may  be 
rudimentary.  Two  pairs  of  antennae.  Abdomen  without 
limbs.  The  females  carrv  the  eggs  in  external  ovi-sacs 
(fig.   138). 


3io 


CRUSTACKA 


To  this  order  belongs  the  freshwater  Cyclops  ;  a  tropical 
species  of  this  genus  is  the  intermediate  host  for  Filaria 
medinensis,  the  well-known  Guinea-worm. 


Fio.  137. — Cyclops  in  various  stages. 


II 


CHAPTER    XVII. 

Pigment  Deposits  and   Degenerations  in   Tissues. 

The  pigment  deposits  are  in  the  main  those  derived 
from  altered  haemoglobin.  Of  these  the  most  important 
is  melanin,  the  residue  from  the  digestion  of  the  red 
corpuscles  by  the  parasites  of  malaria.  This  pigment 
is  taken  up  by  the  leucocytes  and  other  phagocytic  cells 
and  deposited  in  various  parts  of  the  body. 

As  seen  in  the  interior  of  a  parasite  it  is  at  first  finely 
divided  and  varies  in  colour  according  to  the  species 
of  parasite,  later  it  may  be  aggregated  in  each  parasite 
into  a  mass.  In  the  large  masses  which  are  found  in 
the  tissues  it  is  black  with  a  slight  greenish  tinge,  it 
is  insoluble  in  acids,  in  alcohol  and  in  ether,  but  is 
readily  dissolved  by  alkalies.  It  is  very  stable  and  is 
not  destroyed  by  putrefaction.  In  solution  in  alkalies 
it  has  a  distinctly  greenish  tinge,  but  shows  no  charac- 
teristic bands  when  examined  with  the  spectroscope. 
From  this  solution  it  can  be  precipitated  by  the  addition 
of  acids,  and  by  repeated  solution  in  alkaline  fluids 
and  precipitation  with  acids,  can  be  isolated  in  an 
impure  condition.  Analysis  shows  that  it  is  very  rich 
in  iron,  more  so  than  haemoglobin  or  any  of  the  other 
haemoglobin  derivatives.  The  iron  is  in  firm  organic 
combination,  and  does  not  give  the  inorganic  iron 
reactions.  After  frequent  precipitations  it  becomes  brown 
in  colour,  and  the  same  brownish  tinge  can  be  observed 
in  pigment  left  in  certain  organs  as  a  result  of  old 
malarial  invasion. 

Melanin   in  an    acute    attack    of    malaria   is    found   (a) 


312  MELANIN 

in  parasites,  (b)  in  leucocytes,  and  (c)  in  certain  cells  of 
the  connective  tissue  type  in  the  liver,  in  the  parenchy- 
matous cells  in  the  spleen,  and  occasionally  in  the  nuclei 
of  the  endothelium  of  the  capillaries  in  various  parts 
of  the  body,  such  as  the  brain,  liver,  suprarenals,  &c. 
In  an  invasion  of  short  duration  the  only  pigment  found 
is  in  small  granules,  often  no  larger  than  those  set  free 
after  the  breaking  up  of  a  sporulating  body.  At  this 
stage  the  cells  containing  the  pigment  stain  normally 
and  do  not  differ  in  appearance  from  other  cells  of  the 
same  type  which  do  not  contain  pigment. 

When  death  occurs  some  little  time  after  the  termina- 
tion of  an  attack  of  malaria  the  distribution  of  the  melanin 
deposits  is  much  more  limited.  It  will  not  be  found  in 
the  red  corpuscles  as  there  are  no  parasites,  nor  in  the 
leucocytes  or  endothelial  cells,  but  will  be  restricted  to 
the  connective  tissue  cells  in  the  liver  and  the  paren- 
chyma of  the  spleen.  The  pigment  itself  is  now  aggre- 
gated into  larger  masses,  though  these  may  be  seen  to  be 
composed  of  separate  granules.  The  cells  stain  faintly 
with  ordinary  stains  and  often  appear  to  be  shrunken  or 
distorted.  If  examined  still  later  the  pigment  will  only 
be  found  in  blocks  or  masses,  and  the  cells  containing 
these  blocks  will  not  take  basic  stains,  and  appear  merely 
as  an  outline  round  the  pigment  masses,  some  of  which 
appear  to  be  free.  The  change  to  a  brownish  colour, 
particularly  at  the  edges  of  such  masses,  is  sometimes 
to  be  noted. 

Later,  even  months  or  years  after  the  malarial  attack, 
pigment  may  be  found.  If  present  it  will  be  imbedded 
in  the  fibrous  stroma,  and  no  trace  of  the  cells  will  be 
seen.  At  this  stage  the  spleen  is  usually  the  only  organ 
in  which  the  pigment  will  be  found. 

A  consideration  of  these  changes  will  show  that  the 
date  of  a  malarial  invasion  or  invasions  can  be  inferred 
from  the  melanin  deposits  in  the  organs  if  the  state  of 
division  of  the  pigment,  the  staining  reactions  of  the  cells 
containing  the  pigment,  and  the  situation  of  the  pigment 
be  observed. 


HEMOSIDERIN"  313 

It  cannot  be  concluded  because  no  pigment  is  present 
that  there  has  not  been  antecedent  malaria.  We  have 
proof  that  pigment  is  rapidly  removed  from  all  organs 
except  the  liver  and  spleen  (and  occasionally  the  lymphatic 
glands),  and  also  that  it  may  be  removed  from  either  the 
liver  or  spleen.  In  all  recent  attacks  both  organs  contain 
pigment ;  after  a  longer  interval  it  may  be  present  in  the 
spleen  only,  or  rarely  in  the  liver  only. 

In  the  older  cases  when  malarial  infections  have  not 
occurred  for  a  prolonged  period,  the  amount  of  melanin 
found  is  usually  small.  In  some  cases,  even  a  few  weeks 
after  an  attack  of  malaria,  the  amount  of  melanin  is  so 
small  that  careful  search  is  required  to  reveal  it.  It 
seems  probable  that  as  long  as  the  pigment  is  contained 
in  living  cells  it  is  fairly  readily  removed. 

In  many  cases  there  is  evidence  that  the  pigment 
(melanin)  deposited  is  the  result  of  several  distinct 
attacks,  as  in  the  same  specimen  finely  divided  pigment 
in  cells  which  stain  normally,  coarse  pigment  in  cells 
that  stain  poorly,  blocks  of  pigment  with  no  traces  of  a 
containing  cell,  and  pigment  in  between  strands  of 
fibrous  tissue,  can  all  be  seen. 

Melanin  is  the  only  pigment  which  is  characteristic  of 
malaria. 

Another  pigmentary  deposit  of  a  bright  yellow  colour 
is  often  found  in  the  organs  in  cases  of  malaria,  but  this 
is  also  found  in  pernicious  anaemia,  in  the  anaemia  of 
ankylostomiasis,  and  in  other  cases  where  haemolysis  or 
blood  destruction  has  taken  place,  such  as  trypanoso- 
miasis, kala-azar,  and  blackwater  fever,  as  well  as  in  some 
cases  of  poisoning. 

This  yellow  pigment  differs  from  melanin  not  only  in 
colour,  but  in  that  it  is  insoluble  in  alkali  as  well  as 
in  aeid.  It  appears  to  be  slightly  soluble  in  alcohol, 
Whether  or  not  it  contains  iron  is  difficult  to  ascertain, 
as  it  is  frequently  associated  with  other  substances  con- 
taining iron  in  inorganic  combination.  When  found 
alone,  it  usually  does  not  give  the  reactions  for  inorganic 
iron. 


314  IRON    DEPOSITS 

This  yellow  pigment  is  found  in  the  true  hepatic  cells 
in  the  secreting  cells  of  the  kidney,  particularly  in  the 
first  part  of  the  convoluted  tubules,  and  in  the  spleen. 

It  is  evidence  of  blood  destruction  from  any  cause, 
whether  acute,  as  in  blackwater  fever,  or  chronic,  as  in 
pernicious  anaemia  or  ankylostomiasis. 

Both  these  pigment  deposits  can  be  observed  without 
cutting  sections  by  making  "  squash "  preparations  of 
pieces  of  the  organs,  but  the  arrangement  is  better 
shown  in  sections. 

Merely  to  detect  the  pigment,  no  stain  is  needed,  but 
to  show  the  character  of  the  cells  containing  the  pigment, 
it  is  well  to  stain  lightly.  Hematoxylin  gives  good 
results,  but  a  better  stain  is  carmine,  as  both  the  melanin 
and  the  yellow  pigment  stand  out  better  against  the 
red  background.  Thionin  should  not  be  used,  as  it  has 
an  affinity  for  these  pigments  or  the  protoplasm  surround- 
ing them. 

The  method  of  staining  with  carmine  is  as  follows  : — 
Having  removed  the  paraffin  in  the  section  with  xylol, 
the  xylol  with  spirit,  and  the  spirit  with  water,  stain  for 
five  minutes  with  borax  or  lithia  carmine.  Pour  this  off, 
and,  without  washing  with  water,  treat  with  acid  alcohol 
(i  per  cent.)  for  two  minutes.  Remove  this  by  rinsing 
several  times  with  ordinary  spirit,  treat  with  oil  of  cloves, 
to  clear  the  specimen,  and  afterwards  remove  the  oil  of 
cloves  with  xylol,  and  mount  in  Canada  balsam. 

In  many  cases  granules  that  contain  iron  in  a  condi- 
tion to  react  to  the  usual  tests  for  inorganic  iron  are 
associated  with  the  yellow  pigment.  Ammonia  sulphide 
is  sometimes  used  as  the  test  for  the  demonstration 
of  inorganic  iron,  but  has  the  disadvantage  that  the 
brown  sulphide  of  iron  deposited  can  be  confused  with 
malarial  pigment.  A  better  reagent  is  ferro'cyanide  ot 
potassium  in  an  acid  solution,  as  the  blue  ferrocyanide  of 
iron  is  characteristic,  and  causes  no  confusion. 

The  section  should  be  first  treated  with  a  2\  per  cent, 
aqueous    solution    of    potassium    ferrocyanide    for    live 


IRON   DEPOSITS  315 

minutes,  and  then  with  a  1  per  cent,  solution  of  hydro- 
chloric acid  in  glycerine,  or  with  acid  alcohol.  The  acid 
glycerine  should  be  slightly  warmed,  and  must  be  left  on 
till  the  blue  colour  is  quite  distinct.  If  the  blue  colour 
only  shows  faintly,  the  specimen  can  be  replaced  in  the 
ferrocyanide  solution,  and  again  treated  with  acid  glyce- 
rine or  acid  alcohol.  The  specimen  can  then  be  washed 
in  water,  dehydrated  in  alcohol,  cleared  in  xylol,  and 
mounted  in  balsam. 

It  is  important  that  the  sections  should  not  be  touched 
with  iron  after  they  are  cut,  so  that  they  must  not  be 
lifted  with  a  needle,  as  in  any  place  touched  with  iron 
there  may  be  a  deposit  of  the  blue  ferrocyanide  of  iron. 

Loosely  combined  iron  will  be  shown  blue,  whilst  the 
melanin  in  which  the  iron  is  in  firm  combination  will 
remain  black.  The  yellow  pigment  may  be  in  part 
turned  blue  or  may  be  unaltered.  The  outlines  of  the 
cells  can  generally  be  seen  and  counter-staining  is  not 
necessary,  but  weak  carmine  solutions  can  be  used  if 
it  is  desired, 

The  iron  may  be  diffused  throughout  the  cells  or  may 
be  found  in  granules  either  alone  or  mixed  with  yellow 
pigment. 

The  relationship  of  these  ferruginous  granules  to  the 
yellow  pigment  is  not  definitely  known.  In  the  most 
acute  haemolytic  processes,  such  as  in  blackwater  fever, 
both  are  present,  and  the  iron-bearing  granules  are  the 
most  numerous.  In  the  most  chronic  forms,  such  as 
some  cases  of  ankylostomiasis,  yellow  pigment  alone  will 
be  found. 

The  balance  of  evidence  is  in  favour  of  the  view  that 
ferruginous  granules  are  evidence  of  active  and  recent 
haemolysis,  the  remainder  of  the  haemoglobin  being  dis- 
charged into  the  intestine  as  the  iron-free  product  urobilin. 
The  yellow  pigment  is  a  more  permanent  substance,  and 
though  formed  as  a  result  of  acute  haemolysis  is,  when 
in  considerable  amount,  evidence  rather  of  a  prolonged 
or  chronic  haemolysis. 


3*6  PIGMENT 

Some  authorities  hold  other  views,  and  consider  that 
the  yellow  pigment  when  old  gives  the  iron  reaction. 

The  important  point  is  that  both  these  substances 
are  proof  of  blood  destruction,  and  are  not  evidence 
of  malaria,  although  often  found  in  cases  of  malaria. 
They  are  evidence  of  the  general  blood  destruction  that 
may  be  caused  by  the  parasites  of  malaria  as  well  as 
by  other  organisms.  Further  evidence  of  blood  destruc- 
tion is  given  by  the  increase  of  urobilin  in  the  urine  and 
the  much  greater  amount  passed  with  the  faeces  in  acute 
hasmolysis  such  as  blackwater  fever. 

In  the  vicinity  of  certain  skin  lesions  there  may  be 
considerable  disturbance  in  the  normal  arrangement  of 
the  pigment,  so  that  instead  of  being  deposited  only  in 
the  deeper  layers  of  the  epidermis  it  is  scattered  not 
only  in  the  superficial  layers  of  the  epidermis,  but  also 
in  the  subcuticular  connective  tissues. 

This  disturbance  of  the  arrangement  of  pigment  is  most 
conspicuous  in  growths  of  the  granulomatous  group, 
including  lichen  hypertrophicus. 

Pigment  is  normally  present  in  the  skin,  and  it  is  com- 
mon to  find  pigment  in  the  mucous  membranes,  particu- 
larly of  the  mouth  in  the  coloured  races.  Such  pigment 
is  generallv  found  in  patches  in  the  mucous  membrane 
of  the  tongue,  cheeks,  or  gums.  It  has  no  connection 
with  malaria  or  other  disease,  but  is  more  conspicuous 
in  cases  of  advanced  anaemia,  as  the  pigmented  patches 
then  stand  out  more  markedly  against  the  general  white 
background. 

The  normal  pigmentation  of  the  pia  mater  has  been 
already  mentioned.  It  is  extravascular,  and  is  not  dis- 
solved by  alkalies. 

The  pigment  in  all  these  cases  is  much  less  soluble  in 
alkaline  solutions  than  the  melanin  of  malaria.  Pigmen- 
tations of  the  skin  as  a  result  of  Addison's  disease  is  also 
well  known. 

In  melanotic  sarcoma,  black  or  brown  pigment  is  also 
deposited  in  the  growth. 


DEGENERATION  317 

Degeneration.  —  Cells  exposed  to  various  influences 
undergo  degenerative  processes.  Death  or  necrosis  of 
cells  may  take  place,  and  in  such  cases  the  cell  ceases 
to  stain  normally,  so  that  instead  of  taking  up  basic 
stains  it  stains  with  acid  stains,  or  feebly  with  both 
acid  and  basic  stains.  The  nuclei  break  up  and  lose 
their  characteristic  staining  reactions,  and  the  whole 
cell  mav  disintegrate  and  be  converted  into  granular 
debris,  or  caseation  may  take  place  in  which  a  mass 
of  cells  is  replaced  by  granular  fatty  material.  The  mass 
may  become  calcified. 

Where  the  morbid  influences  are  insufficient  to  cause 
cellular  death,  changes  occur  in  the  protoplasm.  Of 
these  the  more  important  are  :  (1)  "Cloudy  swelling,"  in 
which  the  protoplasm  of  the  cell  becomes  swollen  and 
the  aspect  of  the  cell  changed  so  that  its  contents  become 
obscured  and  very  finely  granular.  This  change  is  best 
seen  in  fresh,  unfixed  cells  and  is  shown  in  stained  speci- 
mens by  an  irregularity  in  the  staining.  This  change 
occurs  in  the  early  stages  of  inflammatory  action  and 
may  be  general  in  any  prolonged  pyrexia,  (2)  Fatty 
degeneration  may  affect  any  cells,  but  more  especially 
muscular  fibres  and  the  glandular  cells  of  the  liver, 
kidney,  intestinal  mucosa,  &c. 

In  this  latter  form  droplets  of  fat  are  found  in  the 
interior  of  the  cells  :  these  at  first  are  small,  but  in 
advanced  cases  the  whole  contents  of  the  cell  appear 
to  be  replaced  by  fat  and  the  nucleus  is  squeezed  to  one 
side.  With  fresh  specimens  the  high  refractive  index  of 
the  fat  renders  the  diagnosis  easy.  In  specimens  passed 
through  alcohol,  &c,  the  fat  is  dissolved  out  and  the 
condition  is  then  recognized  by  the  meshwork  of  the 
protoplasm  having  clear,  round,  unstained  spaces  which 
were  previously  occupied  by  the  fat  globules. 

Special  methods  show  this  form  of  degeneration  more 
clearly.  In  specimens  hardened  in  any  of  the  osmic 
acid  fixatives,  such  as  Fleming's  solution,  or  cut  fresh 
and  treated  with  weak  osmic  acid,  the  fat  will  be  stained 


318  FATTY   DEGENERATION 

a  deep  and  intense  black.  Such  sections  can  be  counter- 
stained  with  safranin. 

Soudan   III.  and  Scharlacli   R.  are  also  good  stains  for 

fat.  Fresh  tissues  or  tissues  hardened  in  formalin  must 
be  used.  The  sections  are  treated  with  a  saturated 
alcoholic  solution  of  the  stains  (80  per  cent,  alcohol  for 
fifteen  minutes,  rapidly  rinsed  in  50  per  cent,  alcohol 
and  washed  in  distilled  water.  They  can  be  counter- 
stained  with  hematoxylin  and  mounted  in  any  glycerine 
medium.  The  fat  will  be  stained  a  deep  red.  A  rough 
estimate  of  the  amount  and  extent  of  the  fatty  degenera- 
tion may  be  made  from  such  a  section,  but  the  main 
advantage  of  the  method  is  to  show  the  distribution 
of  the  degeneration  and  the  class  of  cells  mainly 
involved. 

A  promising  method  for  the  estimation  of  the  extent 
of  this  degenerative  process  is  the  determination  of  the 
specific  gravity  of  the  organs.  In  some  cases  the  lat 
is  in  sufficient  amount  to  cause  the  entire  liver  to  float 
in  water,  but  more  commonly  it  is  short  of  this.  To 
determine  the  specific  gravity  a  large  portion  of  an 
organ  is  weighed  and  the  volume  of  this  portion  deter- 
mined. This  volume  can  be  ascertained  in  the  course 
of  an  ordinary  post-mortem  examination  by  the  use  of 
a  vessel  with  an  open  tube  fixed  at  the  side. 

The  vessel  is  rilled  with  water  till  the  water  escapes 
from  the  tube.  When  the  water  has  ceased  to  escape 
a  receiver  is  placed  under  the  tube  and  the  weighed 
portion  of  the  organ  is  placed  in  the  vessel.  Water  will 
again  escape  from  the  tube,  is  collected  in  the  receiver 
and  measured.  The  volume  of  this  water  is  the  same 
as  that  of  the  organ  placed  in  the  vessel,  as  it  is  the 
amount  displaced  by  it. 

We  now  know  the  volume  of  a  given  weight  of  the 
organ  and  therefore  its  specific  gravity.  This  method 
is  sufficiently  exact  for  ordinary  purposes  if  a  sufficiently 
large  piece  of  the  organ  is  taken,  but  for  comparative 
purposes  more  information   is  required  than   we   at   pre- 


AMYLOID   DEGENERATION  319 

sent  possess  as  to  the  normal  variations  in  the  specific 
gravities  of  organs. 

Fatty  degeneration  is  an  important  factor  in  many- 
tropical  diseases.  It  is  marked  in  yellow  fever  almost 
as  much  as  in  poisoning  by  phosphorus.  In  the  anaemia 
of  ankylostomiasis  it  is  constant  and  pronounced,  and 
as  it  affects  extensively  the  intestinal  mucosa,  it  is,  in 
the  more  chronic  cases,  largely  responsible  for  the  im- 
pairment of  the  digestive  processes  in  these  cases.  It 
also  occurs  in  the  liver  and  kidneys  in  this  disease, 
and  as  there  is  also  a  deposit  of  hasmosiderin  these 
organs  often  appear  to  be  of  a  dusky  chrome-yellow 
colour.  The  occurrence  of  this  degeneration  in  the 
cardiac  muscles  is  more  serious,  as  cardiac  failure,  either 
acute  or  chronic,  frequently  is  due  to  this  condition. 

Amyloid  degeneration  is  best  shown  in  fresh  sections. 
Macroscopically  it  can  usually  be  determined  by  treat- 
ing a  cut  surface  of  an  organ  with  tincture  of  iodine; 
a  deep  brown  colour  is  produced  in  such  portions  as 
contain  this  amyloid  material. 

Sections  can  be  similarly  treated  and  mounted  in 
glycerine  media. 

Methyl  violet  stains  amyloid  material  a  deep  red, 
standing  out  clearly  from  the  surrounding  violet. 

Amyloid  degeneration  is  not  common  in  tropical 
diseases,  with  the  exception  of  leprosy.  In  that  disease, 
even  when  there  has  been  no  extensive  suppuration, 
amyloid  degeneration  is  sometimes  found. 

Fibrous  Degeneration.  —  As  a  result  of  degenerative 
changes  in  many  parts,  and  particularly  in  the  nervous 
system,  the  nerve  elements  are  replaced  by  fibrous  tissue. 
This  is  well  seen  in  spinal  diseases  in  which  degenera- 
tion of  nerve  tracts  is  followed  by  the  formation  of 
fibrous  tissue  in  the  tracts  occupied  by  the  degenerated 
nerves.  This  fibrous  tissue  stains  with  ordinary  basic 
stains  and  is  well  shown  by  carmine.  This  change  is 
sclerosis,  and  is  the  final  result  of  the  degenerative  changes. 
The  early  nerve  degenerations  require  special  and  com- 


320  NERVE   DEGENERATION 

plicated  methods  and  could  not  be  satisfactorily  studied 

without  special  knowledge  and  appliances. 

The  simplest  method  for  demonstration  of  early  nerve 
degeneration  is  that  of  Marchi.  Small  pieces  of  tissue 
are  hardened  in  Mailer's  fluid  for  one  week,  taking  care  to 
avoid  mechanical  injury.  The  tissues  are  then  trans- 
ferred to  freshly  prepared  Marchi's  fluid  (Miil lei's  fluid 
two  parts  and  i  per  cent,  aqueous  solution  of  osmic 
acid  one  part)  in  which  they  remain  for  one  week  at 
about  37°  C.  Brain  tissues  require  a  longer  time.  The 
tissues  are  then  washed  for  twenty-four  hours  in  running 
water,  hardened  in  increasing  strength  of  alcohol,  im- 
bedded and  cut.  Sections  are  then  dehydrated,  cleared 
and  mounted  in  balsam.  Nerves  are  best  teased  out  and 
then  mounted. 

Degenerated  nerve  tissue  (fat)  is  stained  black  ;  all  else 
brownish-grey.  The  earliest  degeneration  is  shown  by  a 
flecking  with  black  about  the  internodes  ;  later  fat  drop- 
lets staining  black  more  or  less  completely  replace  the 
white  substance  of  Schwann- Wallerian  degeneration. 


321 


CHAPTER  XVIII. 
Parasites  in  the  Tissues. 

Bacteria,  protozoa,  trematoda,  nematoda,  and  their 
eggs  and  larvae,,  may  be  found  in  the  tissues  in  various 
diseased  conditions  in  man  and  animals  in  various  parts 
of  the  body.     Bacteria  are  considered  separately. 

Protozoa. — The  common  parasites  are  coccidia,  sarco- 
sporidia,  and  flagellates  in  their  resting  stage.  The  first 
two  are  probably  accidental  parasites  in  man,  but  in  other 
mammals  are  very  common. 

Coccidia  are  sporozoa  which  are  encysted,  and  by 
their  continuous  asexual  multiplication  form  visible  white 
masses  not  unlike  tubercles. 

The  rabbit  is  very  commonly  infected,  and  the  large 
masses  are  found  in  the  liver,  though  the  intestinal 
mucosa  may  also  be  infected. 

The  young  coccidia  enter  hepatic  cells  or  those  of 
the  bile  duct  and  speedily  destroy  the  cell.  They  may 
develop  asexually  so  that  a  cyst  is  formed  in  which 
the  protoplasm  divides  into  a  number  of  young  coccidia, 
which,  on  the  rupture  of  the  cyst,  enter  neighbouring 
cells,  and  in  turn  multiply.  Massive  tumours  composed 
of  these  encysted  sporozoa  are  thus  found. 

Some  of  the  young  coccidia  develop  into  sexual  forms, 
male  and  female. 

In  the  male  forms  the  cell  protoplasm  and  nucleus 
divide,  so  that  a  number  of  motile  bodies,  microgametes, 
are  formed.  When  the  cyst  ruptures,  these  microgametes 
enter  a  female  and  fertilize  it.  The  female  forms  macro- 
gametes,  do  not  divide  till  the  microgametes  have  entered 

21 


32  2 


COCCI  IMA 


and  fertilized  them.  After  this  fertilization,  which  lakes 
place  in  the  liver,  the  cvst  wall  of  the  fertilized  macro- 
gametes  becomes  impervious,  and  ultimately  this  cyst, 
now  known  as  the  oocyst,  becomes  detached  and  passed 
with  the  bile  into  the  faeces  of  the  host,  and  is  voided 
with  the  faeces.     Lying  on  the  ground  the  cell  contents  ol 


Fig.   13S. — Coccidia  life  cycle. 


this  oocyst  or  fertilized  macrogamete  divide  so  that  eight 
spores  are  formed.  When  swallowed  by  a  suitable  host 
the  cyst  wall  is  dissolved  off,  and  the  spores  enter  cells 
and  become  young  coccidia  (fig.  138). 

The  different  coccidia  which  have  occurred  in  man 
are  :  — 

Coccidium  cuniculi  (C.  oviforme),  in  which  the  oocysts 


COCCIDIA  323 

measure  '033  to  '049  mm.  long  by  "015  to  '028  mm.  wide. 
This  parasite  has  been  found  in  the  liver  in  man  on 
three  or  four  occasions. 

Coccidium  hominis,  in  which  the  oocysts  measure 
•024  to  "035  mm.  by  *oi2  to  '020  mm.  This  is  a  parasite 
of  the  intestinal  epithelium  of  rabbits  in  which  it  causes 
death  from  severe  diarrhoea.  It  has  been  described  in 
the  intestine  of  man  on  a  few  occasions.  Some  authori- 
ties hold  that  it  is  identical  with  C.  cimiciili. 

Coccidium  bigeminum,  in  which  the  oocysts  measure 
•012  to  '015  mm.  by  "007  to  "oio  mm.  In  this  form 
the  oocyst  divides  into  two  parts,  each  of  which  encysts 
and  forms  sporoblasts.  A  small  number  of  cases  have 
been  described  in  man. 

Demonstrations. — The  demonstration  in  tissues  of  the 
coccidia  is  not  difficult,  as  even  without  staining  the 
shape  and  general  appearance  can  be  readily  made  out. 
They  are  very  liable  to  be  mistaken  for  eggs  of  trema- 
todes.  To  stain  the  cell  contents  is  a  difficult  matter. 
The  wall  of  the  organism  is  not  readily  penetrated  by 
stains,  and  when  the  stain  does  penetrate  the  contents 
are  apt  to  be  so  deeply  stained  that  details  cannot  be 
made  out.  Good  results  can  be  obtained  by  staining 
with  iron  alum — hematoxylin. 

The  method  is  as  follows.    The  solutions  required  are  : — 

A.  2-5  per  cent,  solution  of  iron  alum. 

B.  Hematoxylin  crystals            ...          ...  ...        I  grm. 

Absolute  alcohol        ...          ...          ...  ...  10  c.c. 

Distilled  water            ...          ...          ...  ...  90  c.c. 

This  solution  should  be  kept  for  one  month  to  "ripen."      Then  add  water, 
100  c.c. 

Sections  are  first  placed  in  the  iron  alum  solution  (A) 
for  six  to  twelve  hours,  washed  in  water  for  one  minute, 
then  left  in  the  hematoxylin  solution  (B)  for  twenty- 
four  to  thirty-six  hours.  Afterwards  they  are  washed 
and  differentiated  in  iron  alum  solution  until  the  section 
becomes  deep  blue,  and  the  nuclear  structures  stand  cut 
sharply.     This  stage  is  best   controlled   by  watching   the 


324  SARCOSPORIDIA 

process  under  a  low  power.  When  completed,  wash  in 
running  water  for  fifteen  minutes.  Counterstain  very 
lightly  with  eosin  if  desired.  Dehydrate  and  mount  as 
usual. 

Sarcosporidia. — In  the  muscles  of  some  animals,  such 
as  cattle,  sheep,  pigs,  rats,  &C,  elongated  bodies  are 
found  which  are  often  visible  to  the  naked  eye.  On 
microscopic  examination  these  so-called  "  Mieschers 
tubes,"  or  "  Rainey's  capsules"  are  seen  to  be  composed 
of  a  dense  fibrous  envelope  enclosing  a  vast  number  ot 
spores. 

The  interior  of  the  "Rainey's  capsule"  is  divided  up 
by  extensions  of  the  fibrous  envelope  into  loculi,  each  of 
which  at  an  early  stage  of  growth  contained  a  single  large 
cell  known  as  a  pansporoblast.  Each  of  these  pansporo- 
blasts is  a  single  individual,  only  a  part  of  the  protopla-m 
is  converted  into  spores,  the  remainder  continues  to  live 
and  form  spores.  This  continuous  formation  of  •-pore- 
without  the  destruction  of  the  original  cell  leads  to 
the  formation  of  large  masses.  This  peculiarity  in  the 
asexual  development  separates  the  sarcosporidia  and  the 
allied  forms  which  occur  in  fishes,  Myxosporidia,  from 
the  other  sporozoa,  and  they  are  known  as  Neosporidia  ; 
whilst  the  parasites  in  which  the  whole  protoplasm 
divides  and  the  parental  cell  is  destroyed  in  the  pro- 
cess are  known  as  Telosporidia.  The  sarcosporidia  stain 
readily  with  basic  stains. 

Little  attention  has  been  paid  to  these  bodies.  The 
sarcosporidium  may  be  invisible  to  the  naked  eye,  but 
as  they  usually  produce  some  colour  change,  appear 
as  light  streaks  or  nodules  ;  such  streaks  should  be  looked 
for  and  scrapings  of  them  examined  microscopically.  In 
many  cases  they  are  easily  visible  and  may  be  over  an 
inch  in  length. 

The  nature  of  the  bodies  can  be  demonstrated  in 
sections  or,  better,  by  isolating  one  of  the  bodies  and 
rupturing  it.  A  large  mass  of  spores  is  set  free  and 
these   can  be  readily  stained  after  drying  by  Leishman's 


SARCOSPORIDIA  325 

or  Giemsa's  stain.     They  are  oval,  often   sausage-shaped 

bodies,  with  a  nucleus  with  diffuse  chromatin  staining. 
Often  detached  granules  staining  with  the  polychrome 
red  are  present  in  the  protoplasm.  At  one  end  is  a  clear 
space  which  may  be  unstained  or  very  deeply  stained,  and 
is  known  as  the  polar  capsule.  Nothing  is  known  of  the 
extra-corporeal  life-history  or  sexual  development  ol  s;ir- 
cosporidia.  Similar  bodies  have  been  recorded  in  man 
on  a  few  occasions. 

The  few  species  of  the  sarcosporidia  known  are 
included  in  the  genus  Sarcocystis. 

Another  protozoan  parasite  which  has  been  described 
by  Minchin  and  Fantham  in  man  is  the  Rhinosporidium 
kinealyi.  It  occurs  in  vascular  pedunculated  growths  on 
the  septum  nasi  of  natives  of  India. 

The  youngest  parasites  are  of  irregular  form  and  con- 
sist of  granular  protoplasm  enclosed  by  a  hyaline  mem- 
brane and  containing  numerous  minute  nuclei.  As  the 
parasite  grows  it  becomes  spherical  and  its  hyaline 
envelope  becomes  greatly  thickened,  forming  a  definite 
cyst  wall.  Towards  the  centre  of  the  body  the  proto- 
plasm becomes  segmented  into  spherical  pansporoblasts, 
each  at  first  with  one  nucleus.  In  these  pansporoblasts 
the  nuclei  multiply  and  give  rise  to  spores  numbering 
two  to  twelve.  Each  pansporoblast  thus  becomes  con- 
verted into  a  spore  morula.  Spore  formation  goes  on 
continually  at  the  expense  of  the  peripheral  zone  of 
growing  protoplasm.  A  cyst  will  then  consist  of  three 
zones  within  the  envelope  ;  at  the  periphery  a  zone  of 
uninucleate  pansporoblasts ;  internal  to  this  an  inter- 
mediate zone  of  spore  formation  ;  and  most  centrally 
a  great  number  of  spore-moruLx  (fig.  139).  Nothing  is 
known  of  the  life-history  of  the  parasite  outside  the  body. 

In  dogs  affected  with  rabies  certain  bodies  found  in  the 
brain  may  be  protozoa. 

These,  known  as  Negri  bodies,  occur  mainly  in  the 
grey  matter  of  the  hippocampus  major.  They  are  of 
varying   size,    the   smallest    spherical    and    structureless, 


326 


LEISIIMAX-DONOV.W    BODIES 


larger  oiks  with  a  central  granule  or  nucleus,  whilst  the 
largest  arc  round  or  ovoid  and  contain  several  (as  many 
as   eight)  nuclei  (fig.   140.)     Nothing   is  known   of  their 

development  or  life-history. 


fi^—M 


CX *M^<®§)®;{ 


Fig.  139. — Rhinosporidium  kinealyi.  A,  segment  of  a  section  through 
a  cyst — e,  hyaline  envelope  ;  pz,  pansporoblasts  in  peripheral  zone  ;  iz,  inter- 
mediate zone  of  pansporoblasts  ;  cz,  central  zone  of  spore  morula;.  B.  a  ripe 
spore  morula — sj>,  spores ;  m,  membrane. 


■v^vr^Sr^^^: 


«    - 
Fig.  140.  —Negri  bodies.    ;/,  nucleus  of  nerve  cells  ;  ^,  /i,  the  Negri  bodies. 


The  most  important  of  the  protozoa  found  in  the 
tissues  in  man  are  the  Leishman-Donovan  bodies,  now 
known  to  be  the  resting  stage  of  a  flagellate. 


LEISHMAN-DONOVAN   BODIES  327 

These  bodies  are  found  in  the  spleen,  liver,  mesenteric 
glands,  sometimes  as  shown  by  Cochran  in  superficial 
glands,  submucosa  and  in  the  lungs,  and  are  present  in 
enormous  numbers.  They  are  contained  in  endothelial 
cells,  in  the  macrophages,  and  imbedded  in  a  hyaline 
matrix,  possibly  the  remnant  of  a  broken-down  tissue 
cell  between  the  cells  of  the  organ  or  tissue. 

They  are  small,  round,  oval  or  oat-shaped  bodies, 
characterized  by  having  two  unequal  chromatin  masses, 
one  large  and  oval,  staining  moderately  well  with  the 
red  polychromed  methylene  blue,  and  the  other  smaller, 
more  compact,  usually  rod-shaped,  and  staining  very 
deeply  with  chromatin  stains.  They  may  be  readily 
seen  in  smears  made  from  an  infected  organ  and  can  be 
recognized  in  sections.  During  life  they  may  be  obtained 
by  puncture  of  the  spleen  or  liver.  They  are  more  easily 
obtained  from  the  spleen,  but  there  is  a  certain  amount 
of  risk  in  puncturing  that  organ.  They  are  not  so 
numerous  in  fluid  drawn  from  the  liver,  but  the  risk  in 
puncturing  that  organ  is  slight,  and  therefore  it  is  on  the 
results  of  liver  puncture  that  the  diagnosis  should  be 
made.  The  puncture  must  be  made  with  strict  antiseptic 
precautions,  and  there  must  be  no  fluid  in  the  syringe 
used,  or  the  bodies  will  be  broken  up.  Aspiration  must 
be  slow  and  gradual,  or  blood  will  be  sucked  up  in  too 
great  a  quantity. 

In  the  peripheral  blood  at  some  stages  of  the  disease, 
the  bodies  may  be  found  in  the  leucocytes.  They  are 
said  to  be  particularly  numerous  in  this  situation  just 
before  death. 

Leishman's  stain  is  an  excellent  one  for  smears  of  the 
organs,  or  smears  of  the  fluid  obtained  by  puncturing 
the  liver,  or  to  demonstrate  the  parasites  in  the  leucocytes  ; 
dilute  carbol  fuchsin  also  gives  satisfactory  results. 

In  sections,  carbol  thionin  or  haemalum  used  as 
described  for  malaria  parasites  in  sections  of  the  blood- 
vessels gives  good  results.  Van  Gieson's  stain  brings 
them  out  rather  better  (vide  p.  86). 


328  WORMS 

The  bodies  appear  very  small  in  sections  and  the  two 
chromatin  masses  arc  close  to  each  other,  as  in  the 
hardening  process  there  is  great  shrinking  of  the  parasites. 

The  disease  caused  by  these  parasites  is  that  known  as 
kala-azar.  Similar  bodies  can  be  obtained  by  scraping 
the  floor  of  certain  ulcerated  surfaces  on  the  skin.  These 
ulcers  are  the  Oriental  sores  or  Delhi  boils.  Probably 
the  parasites  are  of  a  different  species,  as  the  topographical 
distribution  of  the  two  diseases  is  different. 

The  two  unequal  chromatin  masses  suggested  to  Leish- 
raan  the  similar  appearances  found  in  degenerate  try- 
panosomes. 

Rogers  and  others  have  shown  that  in  a  medium 
containing  2  per  cent,  citrate  of  soda  changes  occur  in 
the  Leishman-Donovan  bodies  and  that  ultimately  they 
develop  fiagella.  It  is  essential  that  the  temperature  of 
the  culture  should  not  exceed  250  C.  Rogers  states  that 
by  adding  a  little  citric  acid  to  his  cultures,  development 
occurs  more  rapidly  and  certainly;  this  addition  is  not, 
however,  an  essential. 

Helminths. — Trematodes  may  be  present  in  various 
tissues.  Some,  such  as  the  schistosoma,  are  found  in 
blood-vessels;  other  trematodes  may  be  found  in  bile- 
ducts   or  in    the  pulmonary  alveoli. 

Filaria  in  man  and  many  animals  are  found  in  con- 
nective tissue,  or  in  the  muscles,  or  in  the  peritoneal 
cavity.  Others,  as  in  some  cattle,  are  found  beneath  the 
endothelium  of  large  vessels,  such  as  the  aorta,  and  others, 
again,  partly  beneath  the  endothelium  and  partly  free  in 
the  lumen  of  the  vessel. 

Some  nematodes  penetrate  the  intestinal  wall,  ;is  the 
Trichinella  spiralis,  Strongyloides  intestinalis  and  cesopha- 
gostoma  occur  encysted  in  the  submucosa  common  1  v. 
The  Anchylostoma  duodenale  is  reported  to  so  occur  excep- 
tionally. Eggs  are  not  uncommon  in  the  tissues.  Those 
of  Schistosoma  hcematobium  are  commonly  present  in  the 
submucosa  of  the  bladder  or  rectum,  and  of  the  S. 
japonicum    in    the    submucosa    of    the    small     and     large 


WORMS  329 

intestines.  Eggs  of  these  worms  may  be  found  in 
other  parts  and  those  of  S.  japonicum  appear  to  be 
constantly  present  in  the  liver  and  lymphatic  glands, 
whilst  the  ova  of  S.  haematobium  are  sometimes  found  in 
the  lungs. 

Embryos  are  frequently  found  in  blood-vessels  and 
exceptionally  are  extravascular,  but  in  the  natural  life- 
history  of  Trichinella  spiralis  the  larvae  are  encysted  in 
muscles  of  various  parts  of  the  body  and  in  the  dia- 
phragm and  intestinal  walls.  Nematode  embryos,  such 
as  those  of  the  Anchylostoma  duodenale,  penetrate  the 
skin  and  can  be  found  in  the  skin,  subcutaneous  tissues, 
lungs  and  various  parts  of  the  body. 

Eggs  and  larvae  can  be  readily  stained  in  section  with 
any  basic  stain — hematoxylin  and  carmine  perhaps  give 
the  best  results.  They  can  also  be  detected  unstained. 
Larvae  of  Trichinella  spiralis  can  be  well  seen  in  teased- 
out  specimens  or  in  the  intestinal  walls  of  small  animals, 
such  as  rats,  spread  out  between  two  slides.  If  hardened 
whilst  so  spread  out  in  alcohol,  the  intestine  can  then  be 
rapidly  treated  with  sodium  hydrate  solution  till  the 
intestinal  wall  is  rendered  transparent.  The  worms  are 
then  more  readily  seen. 


}o 


CHAPTER    XIX. 

R-ECES. 

The  examination  of  fasces  is  of  the  greatest  import- 
ance, as  most  of  the  intestinal  entozoa  deposit  their  eggs 
whilst  in  the  intestinal  canal;  others  inhabiting  the  liver 
deposit  their  eggs  in  the  biliary  ducts,  while  others,  again, 
pass  their  eggs  or  embryos  through  the  tissues  into  the 
alimentary  canal.  The  eggs  of  all  these  are  passed  with 
the  faeces.  In  other  cases  the  parasites  themselves  may 
be  passed,  or  in  the  case  of  tape-worms  the  mature  seg- 
ments or  proglottides  only. 

In  dysentery,  cholera,  &c,  the  organisms  found  in 
these  diseases  are  present  and  can  be  isolated  from  the 
stools. 

Macroscopic  examination  of  the  stools  is  very  necessary 
and  much  information  can  be  gained.  The  stools  can 
be  examined  as  passed  in  any  vessel,  but  are  more  con- 
veniently examined  if  passed  into  transparent  glass 
vessels  ;  these  can  be  covered  with  a  larger  glass  cover 
fitting  over  the  lower  vessels  like  an  enlarged  Petri  dish. 

The  points  to  observe  are  : — 

(i)  The  presence  or  absence  of  blood,  mucus,  muco- 
pus  or  pus,  and  the  arrangement  relative  to  the  stool  of 
such  a  discharge. 

(2)  The  colour  of  the  stool  and  its  consistence. 

(3)  The  presence  or  absence  of  evidence  of  gaseous 
fermentation. 

(4)  The  odour. 

(5)  The  reaction,  determined  as  soon  as  possible  after 
the  stool  is  passed. 


MUCUS    IN    FAECES  33 1 

(6)  The  bulk  of  the  stools. 

(7)  Any  visible  signs  of  animal  parasites,  sueh  as  the 
worms  themselves  or  the  proglottides  or  segments  of 
tape-worms. 

(1)  Mucus  alone,  or  streaked  or  mixed  with  the  blood, 
usually  indicates  inflammatory  action  in  the  lower  bowel, 
not  necessarily  dysenteric.  It  may  be  caused  by  anything 
that  sets  up  such  inflammation,  such  as  bilharzia,  ulcer- 
ated haemorrhoids,  or  chronic  ulcerations  of  various  kinds 
of  the  rectum.  These  latter  include  malignant  growths, 
granulomatous  growths,  and  the  ulceration  left  as  a 
sequela  of  dysentery.  Rarely  mucus  derived  from  the 
small  intestines  is  passed  with  the  faeces.  Such  mucus  is 
recognized  easily  as  it  is  usually  stained  yellow  with  bile. 

Clear  mucus,  whether  streaked  with  bright  blood  or  not, 
without  any  admixture  of  faecal  matter,  is  met  with  in 
early  or  acute  dysenteric  attacks.  Turbid  or  purulent 
mucus,  sometimes  in  large  quantities,  and  passed  either 
without  any  stool  or  with  solid  formed  motions,  is  more 
indicative  of  a  chronic  ulceration  of  the  rectum,  from 
whatever  cause. 

Sometimes  the  mucus  is  passed  in  large  masses  and 
consolidated,  and  may  include  much  debris  and  numerous 
epithelial  cells.  In  the  condition  known  as  membranous 
colitis,  complete  casts,  several  inches  in  length,  of  the 
rectum  may  be  passed.  These  are  usually  twisted  up 
when  passed,  and  may  be  mistaken  for  worms.  They 
can  sometimes  be  floated  out  in  water,  and  in  any  case 
the  microscopic  structure  should  render  any  mistake 
impossible. 

With  ulceration  limited  to  the  rectum,  stools  are  often 
coated  with  mucus.  The  more  intimately  the  mucus 
and  blood  are  mixed  with  the  faeces,  the  higher  up  are 
the  lesions  from  which  the  mucus  or  blood  is  derived. 
In  some  lesions  the  mucus  is  so  intimately  mixed  with 
the  fluid  fasces  that  it  is  difficult  to  discern,  but  tilting 
the  vessel  from  side  to  side  will  often  indicate  its  pre- 
sence by  the  manner  in  which  the  stool  flows.     In  some 


532  BLOOD    IX    FjECES 

cases  it  is  better  shown  by  adding  water  to  the  faeces, 
when  the  flakes  or  masses  of  mucus  can  be  more  readily 
seen,  especially  if  the  diluted  faeces  are  poured  from  one 
vessel  to  another.  Amccba  arc  killed,  or  have  their 
motility  destroyed  by  this  addition  of  water,  and  there- 
fore this  method  should  only  be  adopted  after  microscopic 
examination. 

Blood  may  be  passed,  bright  red  or  in  clots,  in  large 
quantities.  This  is  no  proof  that  it  is  passed  from  the 
rectum,  as  if  in  sufficient  quantity  and  not  mixed  with 
the  faecal  contents  of  the  intestine  it  need  undergo  very 
little  change  in  passing  through  the  huge  intestine.  Such 
blood  is  occasionally  passed  in  ankylostomiasis.  If 
intimately  mixed  with  the  faeces,  it  may  have  lost  com- 
pletely the  red  colour  and  appear  black  and  tarry — 
melaena. 

When  in  verv  small  amount  altered  blood  can  be 
recognized  by  Weber's  test.  The  fat  is  first  extracted 
with  ether,  and  the  stools  are  then  rubbed  up  with  water 
and  a  third  part  of  acetic  acid  added.  They  are  now 
shaken  up  with  ether  and  an  ethereal  solution  ol  acid 
haematin  is  obtained  if  altered  blood  is  present  {vide 
table  of  spectra). 

In  other  cases,  though  still  red,  the  stool  has  a  duller 
colour,  more  like  anchovy  sauce.  Such  stools  are  passed 
in  some  cases  of  dysentery  where  the  small  intestines  are 
implicated,  and  may  also  be  passed  in  cases  of  extensive 
enteritis  secondary  to  malaria. 

Microscopic  as  well  as  macroscopic  examination  of  the 
mucus  and  blood,  as  well  as  of  the  stool,  should  be  made. 

(2)  The  colour  of  the  stool  is  much  modified  by  the 
diet,  milk  especially  causing  pale  stools.  Articles  of 
diet  taken  by  a  patient  have  a  marked  effect,  and  amongst 
abnormal  articles  that  may  be  nut  with  are  earths  of 
various  kinds,  coal-dust,  &c,  which  to  the  inexperienced 
may  cause  much  confusion.  The  pipe-clay  stools  of 
obstructive  jaundice  may  be  simulated  by  those  of  some 
earth-eaters,  and  the  black  stool  ol  the  coal-dust  eater  has 


BILE   ACIDS   AND    PIGMENTS  333 

been  mistaken  for  melaena.  The  dark  blue  stools  passed 
by  patients  taking  methylene  blue  are  easily  recognized, 
and  so  are  the  bright  yellow  stools  passed  by  patients 
when  taking  ipecacuanha. 

Bile  pigments  and  acids  are  not  usually  present  in 
normal  feces,  as  they  are  absorbed  in  the  small  intestine 
or  broken  down  in  the  large  intestine.  They  may  be 
present  in  diseased  conditions  or  when  the  food  is  passed 
too  rapidly  through  the  intestinal  canal.  Urobilin,  accord- 
ing to  Ross,  is  a  measure  of  the  blood  destruction  taking 
place  in  the  liver. 

Bile  acids  may  be  recognized  by  Pettenkofers  reaction. 
A  small  portion  of  the  fasces  is  mixed  with  a  little  sugar 
and  placed  on  a  white  porcelain  dish.  A  little  sulphuric 
acid  is  allowed  to  come  in  contact,  and  if  bile  acids  are 
present  a  crimson  colour  appears. 

Bile  pigment  may  occur  either  as  Bilirubin  or  as 
Biliverdm. 

(a)  Schmidt's  Reaction. — A  saturated  solution  of  per- 
chloride  of  mercury  is  added  to  the  fasces,  and  in  the 
presence  of  bile  pigments  a  bright  green  colour  is 
produced. 

(b)  Guiclin's  Reaction. — A  drop  of  yellow  nitric  acid 
(i.e.,  containing  nitrous  acid)  is  brought  into  contact  with 
the  fasces,  and  in  the  presence  of  bile  pigment  there  is  a 
play  of  colours,  one  of  which  must  be  green.  This  test 
is  more  decisive  with  a  watery  extract  of  the  fasces. 

(c)  Huppeii's  Test. — The  fasces  are  mixed  with  slaked 
lime  suspended  in  water,  and  the  precipitate  is  filtered 
and  washed.  An  extract  of  the  dried  precipitate  is  made 
with  hot  alcohol  and  a  little  sulphuric  acid.  If  bile 
pigments  are  present  this  extract  is  green. 

Urobilin  is  present  in  small  quantities,  "03 — -o6  gramme 
per  diem  in  normal  stools.  In  cases  of  any  hasmolytic 
disease  the  amount  is  much  increased,  and  is  markedly  so 
in  malaria,  the  increase,  according  to  G.  C.  E.  Simpson,  is 
greater  in  subtertian  malaria,  up  to  173  grammes,  than 
in  benign  tertian.      In  exceptional  cases  it  maybe  much 


334 


ODOIR   AND   REACTION   OF   FAECES 


greater.  Urobilin,  though  il  does  not  contain  iron,  is  one 
of  the  final  products  of  the  destruction  of  haemoglobin. 

Simpson  extracts  the  urobilin  from  the  fasces  by 
repeatedly  shaking  the  faeces  with  large  amounts  of 
water  acidulated  with  dilute  sulphuric  acid. 

The  filtrates  are  then  freely  exposed  to  daylight  for 
some  time  and  examined  spectroscopicallv. 


B     C 


E  b 


Fig.    141.— i,   Spectrum  of  urobilin  ;    2.  spectrum  of  urobilin,  masked  by 
other  pigments. 


The  method  of  quantitative  determination  was  by 
observing  the  amount  of  dilution  required  to  render 
invisible  the  spectrum  of  urobilin  in  a  layer  15  mm. 
thick. 

The  spectroscope  was  standardized  with  a  solution  of 
purified  urobilin  of  known  strength. 

The  consistence  of  the  stool  is  of  great  importance, 
and  it  will  be  found  that  "looseness"  of  stools  is  of  more 
importance  in  tropical  practice  than  in  England.  In 
ulceration  of  the  caecum  and  upper  part  o!  the  colon, 
even  when  this  is  acute  and  extensive,  there  need  be 
neither  visible  mucus  nor  blood,  nor  even  tenesmus. 
"Tropical  diarrhoea"  is  frequently  shown  at  post-mortem 
examinations  to  be  dysenteric.  It  is  very  fatal.  On  the 
other  hand,  mucus  and  blood  may  be  passed  with  formed 
or  even  hard  stools  when  there  are  a  few  chronic  ulcers 
high  up  in  the  large  intestine. 

(3)  In  some  forms  of  tropical   diarrhoea,   particularly 


BULK   OF    EXCRETA  335 

that  form  known  in  the  East  as  sprue,  the  stools  passed 
are  full  of  air-bubbles  and  are  undergoing  active  gaseous 
fermentation. 

(4)  The  odour  varies  so  greatly  with  the  diet  that  it 
is  of  minor  importance.  In  the  races  subsisting  mainly 
on  a  scanty  vegetable  diet  the  odour  is  singularly  slight. 
The  smell  is  mainly  due  to  indol  and  skatol.  In 
cases  of  dysentery  associated  with  formation  of  sloughs 
the  ordinary  faecal  odour  is  replaced  by  the  peculiar  pene- 
trating smell  associated  with  that  condition.  Excessive 
decomposition  of  the  stools  may  cause  an  increase  in 
the  intensity  of  the  normal  smell,  or  if  the  diet  is  mainly 
of  carbohydrate  foodstuff  no  increase  but  even  a 
diminution. 

Variations  in  the  odour  indicate  changes  in  the  decom- 
position of  the  contents  of  the  intestine,  often  from 
variations  in  the  food,  but  sometimes  from  variations  in 
the  "flora"  of  the  intestinal  contents,  rarely  from  struc- 
tural lesions  of  the  intestinal  wall. 

The  result  of  the  administration  of  intestinal  anti- 
septics is  more  often  a  diminution  in  the  putrefactive 
changes  in  the  contents  of  the  bowel  than  any  real 
improvement  in  the  diseased  condition  of  the  intestinal 
wall. 

(5)  The  normal  reaction  of  the  faeces  as  determined  by 
litmus  is  nearly  neutral  ;  when  fasting  it  is  acid,  with  a 
milk  diet  faintly  alkaline.  It  is  usually  acid  to  phenol- 
phthalein.  In  many  cases  of  diarrhoea  and  dysentery  this 
is  replaced  by  a  decidedly  alkaline  reaction.  To  determine 
the  reaction  the  faeces  must  be  examined  as  soon  as  they 
are  passed,  as  a  change  rapidly  occurs  in  most  faeces, 
particularly  when  fluid,  rendering  them  alkaline.  Solid 
motions  must  be  rubbed  up  with  water  in  a  mortar. 

(6)  Bulk  of  Fcvccs. — The  amount  of  faeces  passed  by 
a  European  on  the  average  is  about  130  grammes  per  die  in. 
On  a  meat  diet  it  is  about  half  this.  In  vegetarians  it 
is  much  greater.  The  amount  passed  varies  with  the 
amount    of    food    taken,    and    inversely    as    the    amount 


33,6  ANALYSIS    OF    F.ECES 

digested.  The  excretions  from  the  intestinal  wall  form 
a  proportion  of  the  fasces,  as  during  prolonged  fasts 
22  grammes  may  still  be  passed.  Most  native  races  con- 
sume a  large  amount  of  crude  carbohydrates  in  bulky 
vegetables.  Much  of  this  is  indigestible,  and  is  therefore 
passed  with  the  fasces.  The  average  weight  of  the  excreta 
in  native  races  is  greater  than  in  Europeans,  and  in  India 
is  about  233  grammes.  These  amounts  are  of  importance 
in  estimating  the  amount  of  excrementitious  matter  that 
has  to  be  disposed  of  in  a  community.  It  is  usuallv 
estimated  per  1,000  of  the  population,  and  is  given  for 
a  European  community  as  half  a  ton,  and  for  a  native 
community  as  two-thirds  of  a  ton  (these  figures  including 
urine,  &c). 

The  bulk  of  the  excreta  is  variously  affected  in  disease. 
Discharges  from  the  intestinal  walls,  usually  watery  in 
character,  may  form  an  important  part  or  even,  as  in 
cholera,  nearly  the  whole  of  the  excreta. 

In  other  cases  where  there  is  an  extensive  ulcerated 
surface  muco-pus  may  be  discharged  in  quantity  and 
no  fasces  at  all.  Abscesses  such  as  hepatic  abscesses  may 
open  into  the  intestine,  and  then  there  may  be  a  profuse 
discharge  of  pus,  usually  of  an  anchovy  sauce  colour. 

Water  is  generally  absorbed  in  the  small  intestines,  but 
in  many  conditions  where  the  intestines  are  irritated, 
food  and  even  water  are  hurried  so  quickly  through  the 
alimentary  canal  that  little  absorption  takes  place. 

In  some  diseases,  as  in  pneumonia,  and  towards  the 
crisis  in  relapsing  fever,  there  is  a  great  tendency  to  the 
occurrence  of  frequent  large  watery  motions. 

In  dysentery,  as  a  rule,  though  the  motions  are  frequent, 
the  amount  of  fasces  passed  is  small,  and  in  the  acute 
and  early  stages  no  fasces  at  all  may  be  passed,  though 
mucus  or  blood,  or  both,  are  passed  in  quantity. 

In  most  general  diseases,  as  in  malaria,  digestion  and 
absorption  are  fairly  active,  and  the  motions  then  are 
constipated,  but  the  amount  passed  is  not  more  dimin- 
ished than  might  be  anticipated  from  the  diminution  in 
the  amount  of  food  taken. 


ANALYSIS   OF   FiECES  337 

In  the  disease  known  as  sprue  or  psilosis  the  motions 
are  usually  bulky.  Digestion  and  absorption  are  both 
imperfect.  As  the  guiding  principle  in  the  treatment  of 
this  disease  is  to  give  as  complete  rest  to  the  alimentary 
canal  as  is  consistent  with  a  sufficient  supply  of  nutriment 
to  the  tissues,  it  is  of  importance  to  know  what  forms 
of  food  are  not  digested  and  which  are. 

In  other  diseases,  as  in  obstruction  of  the  pancreatic 
duct,  it  is  also  important  to  know  which  of  the  digestive 
juices  is  wanting,  as  it  may  be  possible  to  supply  the 
deficiency  or  so  modify  the  diet  that  little  or  no  call  for 
this  agent  is  necessary. 

An  analysis  of  the  fasces  will  show  which  of  the 
important  food  elements  have  undergone  little  or  no 
change,  provided  that  the  amount  and  composition  of 
food  taken,  from  which  the  fasces  is  derived,  is  accurately 
known. 

Allowance  has  to  be  made  for  excretions  from  the 
intestinal  wall,  and  for  the  secretions  from  the  liver, 
pancreas,  &c,  which  are  poured  into  the  intestine  in 
variable  amounts. 

The  investigation  is  difficult,  as  the  amount  of  the 
main  ingredients  excreted  in  this  way  may  be  consider- 
able, and  is  affected  by  the  food  taken.  The  amount  of 
fats  excreted,  for  instance,  is  greater  when  fat-free  food 
is  taken  than  in  the  same  individual  when  he  is  fasting. 

For  any  investigation  food  of  known  composition 
and  amount  must  be  taken.  The  simplest  diet  is  a  milk 
diet.  Some  inert,  easily  recognized  substance,  such  as 
charcoal  or  carmine,  should  be  taken  at  the  commence- 
ment of  the  experiment.  The  first  faeces  containing  this 
substance,  usually  twenty-four,  hours  after  the  administra- 
tion, but  in  cases  of  diarrhoea  four  hours,  or  even  less, 
must  be  saved,  as  well  as  all  subsequent  excreta.  A  period 
of  three  days  or  five  days  should  be  taken,  and  at  the 
completion  of  the  experiment  a  second  dose  of  charcoal 
given.  As  soon  as  the  charcoal  appears  in  the  faeces  the 
experiment  is  over. 
22 


33^  ANALYSIS   OF   FjECES 

The  ta?ces  passed  during  the  period  must  be  all 
collected,  and  should  be  analysed  :  (a)  For  water  ;  (6) 
for  nitrogen  ;  (c)  for  fats  ;  (</)  for  carbohydrates  ;  {e) 
urobilin. 

(a)  The  water  can  be  determined  by  weighing  before  and 
after  drying,  but  as  volatile  substances  are  present  this 
must  be  conducted  over  sulphuric  acid  in  a  drying 
chamber  not  over  6o°  C.  The  average  amount  of  water 
is  75  per  cent. 

(b)  Nitrogen  can  be  determined  by  taking  a  weighed 
portion  of  the  faeces,  to  which  is  added  15  or  20  c.c. 
of  x'tj  sulphuric  acid  to  prevent  loss  of  ammonia. 

This  mixture  is  then  dried  in  a  water-bath  till  fairly 
hard,  and  the  desiccation  completed  in  a  drying  chamber 
at  io°  C.  over  sulphuric  acid. 

A  gramme  of  this  dried  powder  is  then  mixed  with  25  c.c. 
of  strong  sulphuric  acid  and  1  grm.  of  sodium  pyro- 
phosphate, and  this  is  allowed  to  stand  for  some  hours, 
and  then  cautiously  boiled.  The  nitrogen  will  all  now 
be  in  the  form  of  ammonium  sulphate. 

This  can  be  estimated  after  allowing  to  cool  by  adding 
600  c.c.  of  water  and  sodium  hydrate  solution  tiil  strongly 
alkaline.  Some  granulated  zinc  to  prevent  bumping 
should  also  be  added. 

The  mixture  is  then  distilled  and  the  distillate  allowed 
to  pass  into  a  measured  amount  of  $j  sulphuric  acid. 
The  ammonia  will  neutralize  a  certain  amount  of  this, 
so  that  by  subsequent  titration  the  amount  of  ammonia 
that  has  distilled  over  can  be  estimated. 

Undigested  proteids  passed  unchanged  form  a  very 
small  part  of  the  nitrogen. 

(c)  Fats  are  usually  determined  as  "  total  fats,"  consist- 
ing of  fats,  soaps  and  fatty  acids. 

The  faeces  should  be  thoroughly  dried  over  sulphuric 
acid  and  treated  with  1  per  cent,  hydrochloric  acid  to 
split  up  the  soaps. 

Ether  is  then  added  and  the  ethereal  extract  after  drying 
is  re-dissolved  in  water-free  ether. 


PARASITES  339 

The  residue  left  after  the  evaporation  of  the  ether  is 
considered  as  total  fats. 

In  any  question  of  the  digestion  of  fats  it  must  be 
remembered  that  fats  differ  greatly  in  digestibility,  and 
that  a  healthy  person  will  pass  unchanged  about  8  per 
cent,  of  mutton  suet,  which  melts  at  520  C,  whilst  he 
will  only  pass  2*5  per  cent,  of  pork  fat,  which  melts  at 
300  C.  A  healthy  adult  on  a  pure  milk  diet  will  digest 
about  95  per  cent. 

(d)  Sugar  and  carbohydrates  are  usually  digested  com- 
pletely unless  enclosed  in  an  impervious  capsule,  as  in 
some  vegetable  foods. 

The  amount  of  gas  formed  from  such  faeces,  kept  at 
blood-heat  for  twenty-four  hours,  will  give  a  fair  indication 
of  the  amount  of  carbohydrates  present. 

(6')  Urobilin  to  indicate  the  amount  of  blood  destruc- 
tion taking  place. 

Parasites. 

(7)  Parasites  of  various  kinds  may  be  seen  by  direct 
examination,  but  more  often  it  is  necessary  to  strain 
the  stools.  This  is  best  done  by  placing  the  stool  on 
a  muslin  or  strong  fine  wire  gauze  strainer  (fig.  142)  and 
adding  water  and  stirring  well.  By  repeating  this  process 
all  the  smaller  particles  of  the  fasces  will  be  carried 
through  the  muslin,  and  only  the  coarser  particles  and 
any  entozoa  present  will  be  left  on  the  strainer.  Some 
of  the  smaller  entozoa  may  be  carried  through  the 
strainer.  The  fluids  that  have  passed  through  can  be 
strained  again,  or  passed  through  a  muslin  bag.  This  is 
conveniently  clone  with  a  bucket  big  enough  to  hold 
the  sieve.  The  bucket  is  filled  with  water  so  that  it 
covers  the  wire  gauze.  The  faeces  are  then  placed  on 
the  gauze  and  stirred  well.  From  time  to  time  the  sieve 
is  lifted  so  that  the  stirred  faeces  pass  into  the  bucket. 
When  the  water  becomes  turbid  the  strainer  is  placed  in 
a  second  bucket  filled  with  clear  water  and  the  process 
repeated.     On  examining  the  strainer  the  greater  number 


340 


MICROSCOPIC    EXAMINATION' 


of  the  worms  will  now  be  readily  seen.  After  standing 
a  little  the  superjacent  fluid  in  both  buckets  should  be 
poured  off  and  the  deposit  again  passed  through  the  sieve. 
More  worms  will  be  found.  Entozoa  are  damaged  a  little 
by  this  proceeding.  When  they  are  required  for  detailed 
examination  they  should  be  picked  out  of  the  undiluted 
faeces. 


Fig.  142.— Wire  Gauze  Strainer. 


Microscopic  Examination. — The  most  important  objects 
of  this  are  (1)  the  detection  of  ova  of  parasites,  (2)  the 
detection  of  animal  micro-parasites,  and  (3)  the  investiga- 
tion of  the  bacteria  present. 

Ova  are  readily  seen  with  a  low  power,  two-thirds  inch 
objective,  but  for  their  identification  at  least  half  an  inch 
or,  better,  quarter  of  an  inch  objectives  are  requisite. 

The  preparation  of  the  stool  is  very  simple.  A  small 
particle  of  the  faeces  is  placed  on  a  slide  :  it  can  be  con- 
veniently taken  up  with  a  splinter  of  wood  such  as  a 
match  stick.  If  not  too  hard  it  should  then  be  com- 
pressed by  a  cover-glass  into  a  thin  layer;  if  too  hard  for 
this  it  can  be  mixed  with  a  little  water.  If  the  stool  be 
watery  it  should  be  allowed  to  stand,  and  with  a  pipette 
some  of  the  fluid  taken  from  the  bottom,  as  the  eggs  are 
heavier  than  the  fluid  stool  and  sink  to  the  bottom  of  the 


EGGS  341 

vessel  containing  it.  The  eggs  that  may  be  met  are  those 
of  the  Ascaris  lumbricoides,  Trichocephalus  dispar,  Aiikylo- 
stomum  duodenale,  Oxyuris  vermicularis,  several  species  of 
tape-worms,  several  species  of  Fasciolidce,  those  of  Schis- 
tosomum  haematobium  with  the  lateral  spine  sometimes, 
and  those  of  S.  japonicum,  and  the  embryos  of  Strongy- 
loidcs  intestinalis.  The  attached  diagram  shows  the 
appearance  of  the  more  important  of  the  ova. 

The  eggs  of  Ascaris  lumbricoides  (round-worm)  are 
enclosed  in  a  thick,  clear  capsule  usually  coated  with  an 
albuminoid  covering  stained  yellow  or  brown  by  the 
faecal  colouring  matter.  The  protoplasmic  contents  are 
granular  and  do  not  as  a  rule  completely  fill  the  inner 
capsule  (fig.  143,  a). 

If  too  much  pressure  has  been  used  the  albuminoid 
covering  may  have  been  ruptured  and  the  egg  is  seen 
surrounded  only  by  its  thick  transparent  capsule.  These 
Ascaris  eggs  can  be  readily  distinguished  from  eggs  with 
thin  capsules,  such  as  those  of  A.  duodenale,  not  only 
by  the  thickness  of  the  capsule,  but  by  its  more  spherical 
shape,  and  by  the  granular  and  unsegmented  character 
of  the  egg  contents.  Unfertilized  eggs  are  larger,  more 
oval,  and  the  egg  contents  contain  numerous  retractile 
globules,  which  should  not  be  confused  with  segmen- 
tation. 

The  eggs  of  Trichocephalus  dispar  (whip-worms)  are 
easily  distinguished,  as  they  are  small  oval  eggs  contained 
in  a  thick,  deeply  stained  outer  capsule  which  has  an 
opening  at  each  end.  Inside  this  capsule  is  a  thinner, 
unstained  capsule,  and  the  egg  contents  are  granular. 
In  many  instances  the  openings  in  the  outer  capsule  are 
seen  to  be  plugged  by  mucus  (fig.  143,  b). 

The  ovum  of  Aukylostoinum  duodenale  is  enclosed  in 
a  single,  thin,  transparent,  unstained  capsule.  At  the  time 
the  egg  is  passed  segmentation  usually  into  about  four 
segments  has  taken  place  (fig.  143,  c1,  c2),  but  if  the  stool 
be  kept  a  large  number  of  segments  will  be  present 
according  to  the  time  and  temperature,  and  in  twenty-four 


Fig.  143. — a,  Ascaris  lumbricoides ;  b,  Triehocephalus  dispar ;  c,  Oxyuris 
vermicularis  ;  c',  r,  Ankylostomum  Juodenale ;  d,  Oncosphere  of  Cesioie; 
e,f,g,  various  Fasciolida ;  h,  Schistosomum  (?)  hcematobium  (from  face*)  ; 
i,  Schistosomum  lue  mat  obi  it  in  (from  urine). 


eggs  343 

to  forty-eight  hours  ;i  fairly  well  formed  embryo  will  be 
found  in  many  of  the  egg  capsules,  and  the  egg  then 
closely  resembles  that  of  Oxyuris  vermicularis.  The  eggs 
of  the  closely  related  Necator  americanus  are  slightly  larger, 
but  otherwise  similar,  to  those  of  the  Ankylostome. 

Oxyuris  vermicularis  (thread-worm)  has  an  egg  that 
in  size  and  general  appearance  is  not  unlike  the  anky- 
lostome, but  is  usually  flattened  at  one  side.  At  the  time 
the  stool  is  passed,  this  egg  contains  a  well-formed 
embryo  (fig.  143,  c). 

The  "eggs,"  or  rather  oncospheres,  of  the  different  species 
of  tape-worms  may  only  present  slight  differences  from 
each  other,  but  they  are  readily  distinguished  from  all 
other  ova  by  the  radial  striation  of  the  thick  capsule  and 
the  presence  of  a  differentiation  in  the  contents  into  an 
embryo  ;  the  six  hooklets  of  this  embryo  can  usually  be 
made  out  (fig.  143,  d).  These  oncospheres  consist  of 
the  embryo  and  embryonic  capsule  only,  the  outer  part 
of  the  egg  having  usually  disappeared. 

The  eggs  of  the  various  Fasciolidce  (flukes)  can  be 
recognized  by  the  presence  of  an  operculum  or  lid 
(fig.  143,  e,  f,  g),  and  distinguished  from  each  other  by 
their  size.  The  eggs  of  the  Trematodes  found  in  man  are 
usually  yellow  or  brown. 

Schistosomum  haematobium  has  a  highly  characteristic 
egg,  as  it  is  armed  with  a  sharp  spike.  In  eggs  passed 
with  the  faeces,  with  which  they  may  be  mixed,  or  con- 
tained only  in  mucus  on  the  surface  of  the  stool,  this 
spike  is  at  one  side  (fig.  143,  //),  in  urine  the  spine  is 
terminal  (fig.  143,  i),  and  by  some  it  is  believed  that  the 
eggs  with  a  lateral  spine  are  from  different  species  of 
Schistosoma.  If  water  be  added  to  the  faeces,  it  will  be 
seen  that  the  egg  contains  a  ciliated  embryo  which  soon 
becomes  active  and  bursts  through  the  egg  capsule.  The 
free-swimming  embryo  remains  alive  in  water  for  some 
days,  but  undergoes  little  further  change.  An  intermediate 
host,  perhaps  a  fresh-water  mollusc,  is  probably  necessary 
for  its  further  development. 


344 


EGGS   AND   WORMS 


Schistosomum  japonicum. — The  eggs  are  passed  into  the 
intestinal  canal  higher  up  than  those  of  S.  haematobium. 
They  have  a  lateral  knob  as  shown  by  Leiper,  in  place  of 
a  spine,  no  operculum,  and  are  about  the  same  size  as  the 
eggs  of  ankylostomes.  They  contain  a  formed  miracidium 
when  passed. 

Measurements  of  Ova. 

Titnia  saginata 
7'esnia  solium 
Hymenolepis  nana 
Dibothriocephalus  latus     ... 
Fasciola  hepatica  ... 
Fasciolopsis  buski 
Dicroccelium  lanceaium     ... 
Clonorchis  sinensis 
Heterophyes  heterophyes    ... 
Paragonimus  westermani . . . 
Gastrodiscus  hominis 
Cladorchis  watsoni 
Schistosomum    hcematobium 
Schistosomum  japonicum  . . . 
Ascaris  lumbricoides 
Oxyuris  vermicularis 
Ankylostomum  duodenale... 
Necator  amei'icanus 
Trichocephalus  dispar 

In  measuring  eggs  no  pressure  must  be  used.  Some, 
such  as  those  of  S.  japonicum,  are  very  readily  distorted 
by  pressure.  Hence  the  variation  in  the  measurements 
given. 

The  embryo  of  the  Strongyloides  intestinalis  is  fre- 
quently passed  with  the  stools.  The  embryos  of  the 
Trichina  spiralis  are  very  rarely  passed  in  the  stools,  as 
they  normally  penetrate  the  intestinal  walls  and  pass 
into  the  surrounding  tissues. 

In  the  faeces,  thread-worms,  segments  of  tape-worms, 
and  occasionally  round-worms  are  passed  naturally. 
After  the  administration  of  powerful  anthelmintics,  the 
whole  tape-worm,  round-worms,  ankylostomes,  flukes, 
and  whip-worms  may  be  passed.  Some  species  are  never 
found  under  any  circumstances  in  the  faeces. 


0*03  to  0*04  mm.  by  002  to  0*03  mm. 

(Spherical)  0*03  mm. 

,, 

0-4      .. 

0*068  to 

0-071  mm.  by  0045  mm.  (operculated) 

0-13  by 

o-o8  mm. 

0*125  „ 

077     », 

0-04     ,, 

0*03     ,, 

0-027  >> 

o-oi6  ,, 

°"°3     .. 

0-017   >! 

o#o8    to 

o-i  mm.  by  0*052  to  0*075  nim. 

0-15    by 

0*07  mm. 

0"I2      ,, 

0.075  .. 

O'OI      ,, 

0.03     „ 

0*06    to 

0.09    ,,  by  0.03     to  0.05  mm. 

0-05     ,, 

0*07     „    ,,  0*04     ,,  0*05  mm. 

0-05    by 

0.016  ,,  to  0*024  mm. 

0*056  to  0.61     ,,  by  0-034  to  0*038  mm. 

0*064  » 

0*072  ,,  ,,  0*036  mm. 

0-05     „ 

0*054  ,,    „  0-023     ,, 

345 


CHAPTER  XX. 

Intestinal  Parasites. 

The  worms  met  with  in  the  human  intestine  and  its 
appendages  belong  to  the  following  classes  : — 

A.  Cestoda. — These  flattened  worms  have  a  segmented 
body,  no  digestive  tube,  and  are  hermaphroditic. 

B.  Trematoda. — In  these  the  digestive  tube  is  incom- 
plete ;  there  is  no  anus,  and  the  body  is  not  segmented. 
They  are  hermaphroditic  except  the  Schistosoma. 

C.  Nematoda. — These  usually  have  a  complete  diges- 
tive tube.  They  are  cylindrical  worms,  and  they  are  not 
hermaphroditic. 

A.  Cestodes. 

The  human  Cestodes  are  :  Tcenia  solium,  T.  saginata, 
T.  confusa,  T.  africana,  Dipylidium  caninum,  Hymeno  lepis 
miirina  (T.  nana),  and  H.  dimimita,  Davainea  madagas- 
cariensis,  Bothriocephaliis  latus,  Diplogonoporus  grandis. 

Cestodes  or  Tapeworms. — The  embryonic  or  cystic  forms 
of  the  Tcenia  echinococcus  may  be  found  in  the  liver, 
muscles  of  man,  &c.  The  definitive  host  is  the  dog. 
These  cysts,  the  hydatid  cysts,  can  hardly  be  mistaken 
for  non-parasitic  cysts  ;  they  can  be  readily  distinguished 
if  there  is  any  doubt  by  the  laminated  cyst  wall  and  the 
presence  of  hooklets  in  the  cyst  or  discharges. 

In  the  case  of  the  echinococcus  man  is  the  intermediate 
host.  A  larval  form  of  Bothriocephaliis  (B.  mansoni)  has 
been  found  in  the  connective  tissues  of  men  in  Japan, 
and  similar  larval  forms  have  been  obtained  from  an 
aboriginal  of  British  Guiana  and  in  Central  Africa.  These 
larvae  may  be  the  larval  form  of  a  DibotJiriocephalus,  but 
it  is  not  certain.  They  are  classed  as  a  separate  genus, 
Sparganum  (Stiles). 


34-6  TAPE-WORMS 

The  greater  number  of  the  tape-worms  found  in  man 
attain  sexual  maturity  in  him.  Man  is  therefore  the 
definitive  host  of  these  worms. 

The  general  structure  of  tape-worms  should  be  known, 
and  the  differences  indicated  in  the  tabular  statement  of  the 
well-known  human  tape-worm  will  then  be  understood. 

Tape-worms  consist  of  a  head  or  fixed  portion  attached 
by  hooks  or  suckers,  or  both,  to  the  intestinal  wall.  This 
"head"  is  called  the  scolex.  From  this  scolex  growth 
takes  place  continuously  in  one  direction  ;  at  first  as  a 
narrow  neck  which  is  not  segmented,  but  which  rapidly 
becomes  segmented,  and  as  growth  continues  each  seg- 
ment increases  in  size  and  becomes  sexually  mature. 
Each  segment  is  known  as  a  proglottis,  and  together 
these  form  the  strobila.  When  sexually  mature,  the  eggs 
are  fertilized,  and  finally  the  genital  organs  atrophy  and 
the  proglottis  is  reduced  to  a  muscular  sac  distended  by 
a  uterus  filled  with  fertilized  eggs.  These  proglottides 
become  detached  and  are  passed  in  the  stool.  Each 
proglottis  is  motile  and  may  live  for  some  time  after  it 
has  Been  passed  in  the  stool.  It  creeps  about  discharging 
its  eggs.  These  eggs  are  taken  up  by  the  intermediate 
host,  another  mammal,  a  fish,  or  even  an  insect,  and 
develop  in  that  animal  into  the  cystic  or  larval  stage. 
In  the  case  of  some  of  the  tape-worms,  as  in  Botlirio- 
ceplialus,  a  ciliated  embryo  is  formed  which  swims  freely 
in  water,  and  in  its  intermediate  host  does  not  form 
a  cyst,  but  an  elongated,  worm-like  larva  known  as  a 
"  plerocercoid"  larva. 

If  taken,  with  food  or  otherwise,  into  the  intestinal 
tract  of  man,  the  cyst  is  set  free  and  the  head  becomes 
the  scolex  of  the  mature  tape-worm.  This  scolex  fixes 
itself  to  the  intestinal  wall  and  gives  rise  to  the  pro- 
glottides by  growth  from  it. 

The  tape- worm  derives  its  nutriment  by  osmosis  from 
the  intestinal  tract.  There  is  no  intestine  and  no  trace 
of  one.  There  are  water  vascular  tubes,  the  water 
vascular  system,  running  the  whole  length  of  the  worm. 


STRUCTURE    OF   TAPE-WORMS  347 

With  this  exception,  and  the  nervous  system,  each  seg- 
ment or  proglottis  is  a  distinct  individual  jointed  on  to 
its  predecessor  and  successor. 

The  points  in  the  structure  of  a  proglottis  are  best 
observed  in  a  half-grown  proglottis,  as  earlier  the  organs 
are  not  fully  developed  and  the  last  segments  are  merely 
muscular  egg-sacs  with  atrophied  organs. 

For  permanent  specimens  the  method  to  be  adopted 
is  as  follows  :  Stain  for  twenty-four  hours  with  very 
weak  borax  carmine  ;  soak  in  glycerine  for  some  months. 
Compress  between  two  slides  clamped  together  and  place 
in  methylated  spirit.  When  partially  hardened  the 
pressure  can  be  relaxed  and  the  specimen  dehydrated 
in  alcohol.  Clear  with  oil  of  cloves  and  mount  in  balsam- 
Pressure  should  be  applied  to  the  cover-glass  till  the 
balsam  has  hardened. 

The  proglottis  is  covered  with  a  transparent  cuticle, 
and  has  a  powerful  muscular  wall  with  longitudinal  and 
transverse  or  circular  bands.  In  the  interior  of  the  seg- 
ment are  the  organs  of  generation,  male  and  female,  as 
each  segment  is  hermaphroditic.  The  arrangement  of 
these  organs  varies  greatly  in  different  species,  but  they 
conform  to  a  common  type. 

The  space  between  the  organs  is  occupied  by  paren- 
chymatous tissue,  in  which  are  often  included  highly 
refractile  calcareous  masses,  which  must  not  be  mistaken 
for  eggs. 

The  male  genital  organs  consist  of  a  number  of  small 
testes.  Minute  vasa  efferentia  unite  about  the  centre  of 
the  segment  into  a  common  vas  deferens,  this  terminates 
in  the  copulatory  organ  or  cirrhits,  opening  with  the 
vagina  into  a  genital  cloaca. 

The  female  genital  organs  consist  of  the  vagina  leading 
as  a  straight  tube  from  the  genital  cloaca  into  an  enlarge- 
ment, the  receptaculum  seminis ;  from  this  the  tube 
is  continued  to  the  shell  gland,  and  near  it  the  ovarian 
tube,  or  tubes,  if,  as  is  usual,  the  ovary  is  paired,  open. 
There  is  a  diverticulum   running   longitudinally   in   the 


W8 


STRUCTURE   OK   TAPE-WORMS 


centre  of  the  proglottis,  which  at  first  is  simple,  but  later 
branched — the  uterus.  The  continuation  of  the  vagina 
is  surrounded  by  the  shell  gland  and  the  duet  of  the 
vitellarium  or  yolk  gland  opens  into  it. 

The    spermatozoa   pass    up    the    vagina  and    the    eggs 
discharged  from  the   ovaries  are  fertilized,  receive  their 


Fig.  144. — a,  Testes  ;  b,  vasa  efferentia  ;  c,  vas  deferens ;  d,  genital  pore  ; 
e,  vagina ;  /,  receptaculum  seminis ;  g,  uterus  ;  h,  shell  gland  ;  ?',  ovary  ; 
j,  vitellarium  or  yolk  glands  ;  w.v.s.,  water  vascular  system. 


yolk  and  shell,  and  are  then  forced  into  the  longitudinal 
diverticulum  or  uterus.  As  more  and  more  eggs  pass 
into  the  uterus  this  tube  becomes  distended  and  the 
lateral  diverticula  enlarged,  and  ultimately  the  whole 
proglottis  is  occupied  by  the  uterus  distended  with  ova. 


CLASSIFICATION    OF   TAPE-WORMS 


349 


The  projection  marking  the  genital  cloaca,  into  which 
both  the  male  and  female  organs  open,  is  known  as  the 
genital  pore  (fig.  144). 

In  examining  a  tape-worm  the  points  to  observe  are  : — 

(1)  The  size,  shape  and  number  of  proglottides  in  the 
worm. 

(2)  The  size  of  the  scolex  and  its  armature,  which  may 
be  suckers  only,  or  suckers  and  hooks,  and  the  number 
of  these. 


Fig.  145. — a,  T<znia  saginata  ;  b,  Dipylidium  ;  c,  Davainea  ;  d,  Hymeno 
lepis  ;  e,  Bothriocephalus  ;  f,  Diplogonoporus  grandis. 

(3)  In  the  proglottides  the  relative  length  and  breadth 
of  the  segments,  particularly  of  the  mature  ones.  The 
number  of  genital  pores  in  each  proglottis — two  in  Dipy- 
lidium and  Diplogonoporus,  one  in  most  of  the  other 
genera.  The  position  of  the  pore,  which  is  marginal  in 
most,  but  in  the  mid-ventral  line  in  the  middle  of  the 
broad  surface  of  the  proglottis  in  Bothriocephalus  and 
on  the  flat  surface  in  all  the  Dibothriocephaloidea.  It 
must  also  be  noted  if  the  genital  pores  in  the  different 


35°  FLUKES 

segments  are  ail  on  the  same  side  of  the  worm,  as  in 
the  Davainea,  or  alternately  (frequently  irregularly  so) 
on  opposite  sides  of  the  worm,  as  in  Tcenia  saginata 
(rig.  145,  a).  In  the  ripe  proglottides  the  branching  of  the 
uterus  should  be  noted  and  the  arrangement  of  the  eggs. 

With  proper  attention  to  these  points  there  is  little 
difficulty  in  differentiating  between  the  different  species 
of  the  human  Cestodes. 

(4)  The  number  of  testes,  and  the  arrangement  of  the 
eggs  in  the  uterus  or  in  the  parenchyma.  The  presence 
of  the  second  opening,  that  of  the  uterus,  must  be  noted 
in  the  Dibothriocephaloidea. 

It  will  be  seen  that  the  dog  is  the  definitive  host  of 
several  species  of  the  human  tape-worms.  Of  the  Dipy- 
lidium  the  dog  is  the  usual  host.  Some  of  the  other 
tape-worms  of  the  dog  have  been  found  in  man.  The 
most  important  cystic  Cestode  of  man,  the  cchinococcus, 
passes  its  adult  stage  in  the  intestine  of  the  dog.  It  is 
therefore  important  to  have  some  knowledge  of  the 
canine  tape-worms.  The  subjoined  table  by  Henry  B. 
Ward  gives  the  leading  characteristics  of  the  best  known 
of  these. 

B.  Trematodes. 

Trematodes,  or  flukes,  are  rarely  met  with  in  man  out- 
side the  Tropics  and  sub-tropical  regions.  Of  the  human 
Trematodes  one,  Paragonimus  westermani  (Distoma  pul- 
monale), is  found  in  the  lungs  ;  others,  the  Schistosomum 
hcematobium  (Bilharzia  hcematobium)  and  S.  japonicum, 
occur  in  the  blood-vessels.  Other  trematodes  are  present 
in  the  liver,  and  the  eggs  only,  which  are  passed  down 
the  bile  ducts,  are  found  in  the  faeces,  and  still  others 
are  found  in  the  intestinal  tract,  so  that  ordinarily  the 
eggs,  and  after  the  administration  of  powerful  anthel- 
mintics, like  thymol,  the  adults  also  of  these  are  passed 
by  the  rectum. 

The  Trematodes  include  the  Fasciolidce,  which  are 
flattened  bodies,  of  oval  shape  with  pointed  ends  ;   from 


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352  STRUCTURE    OF    FLUKES 

the  peculiarity  of  their  shape  they  are  popularly  known 
as  "  flukes."  They  are  hermaphroditic,  non-segmented, 
and  possess  an  incomplete  intestine.  They  are  armed 
with  two  suckers  placed  near  each  other  in  most  of  the 
genera.  One  of  these,  the  oral  sucker,  surrounds  the 
mouth,  the  other,  the  ventral  sucker,  or  acetabulum,  is  on 
the  ventral  surface. 

In  the  Parampliistomida5  the  suckers  are  at  opposite 
ends  of  the  body,  and  they  differ  in  shape  from  the 
other  Trematodes. 

The  intestinal  system  of  the  Trematodes  consists  of 
a  short  muscular  pharynx  leading  from  the  anterior 
sucker  longitudinally.  The  oesophagus  terminates  by 
bifurcating  into  the  two  caeca  which  pass  round  the  body 
towards  the  posterior  extremity  of  the  worm.  These 
caeca  end  blindly,  but  are  often  sacculated  or  have 
diverticula.  The  genital  organs  are  complicated  and  the 
arrangement  varies.  In  Schistosomidce  the  male  and 
female  are  distinct,  and  the  female  lives  in  an  incomplete 
canal,  the  gynaecophoric  canal  in  the  male.  In  the  other 
Trematodes  the  male  and  female  organs  are  contained 
in  the  same  animal",  but  the  openings  of  each  are  distinct. 

The  female  organs  consist  of  a  convoluted  uterus  open- 
ing externally  near  the  second  or  ventral  sucker  in  the 
Fasciolidce.  This  convoluted  uterus  leads  to  a  dilatation 
surrounded  by  the  "shell  gland,"  and  into  this  the  ovarian 
tube  from  the  single  ovary  opens,  and  also  the  opening 
from  the  spermatheca.  The  common  vitelline  duct  formed 
by  the  junction  of  the  two  vitelline  ducts  which  receive 
the  yolk  from  the  numerous  yolk  glands  distributed  along 
the  edges  of  the  animals  opens  with  it.  There  is  a  canal 
leading  from  the  ventral  surface  to  the  oviduct  known 
as  the  canal  of  Laurer,  which  may  serve  for  the 
entrance  of  spermatozoa. 

There  are  two  compound  testicles  which  lie  one  in 
front  of  the  other. 

The  ducts,  vasa  deferentia,  from  these  pass  forwards 
and  open  into  a  dilatation,  the  vesicula  semiualis,  the  duct 


STRUCTURE   OF   FLUKES 


353 


from  which  leads  to  the  penis,  which   opens  externally 
close  to  the  female  genital  opening  (fig.  146). 

The  details  of  the  arrangement  vary  greatly.  Fertiliza- 
tion is  probably  by  a  different  worm.  The  fertilized 
eggs  are  passed  with  the  fasces,  sputum,  urine,  &c,  of 
the  definitive  host. 


Fig.  146. — a,  Anterior  sucker ;  b,  caecum  ;  c,  ventral  sucker  or  acetabulum  ; 
d,  opening  of  uterus ;  e,  yolk  glands  ;  f,  vitelline  ducts  ;  g,  ootype  ;  h,  ovary  ; 
7,  compound  testicles  ;  j,  vesicula  seminalis  ;  k,  penis. 

The  structure  of  Fasciolidce  is  best  shown  as  in 
the  case  of  the  Cestodes  by  prolonged  immersion  in 
glycerine  and  then  passing  through  alcohol  and  oil  of 
cloves,  after  staining  with  weak  borax  carmine  for  some 
days. 

23 


354  CLASSIFICATION    OK    FLUKES 

In  one  division  of  the  Trematoda,  the  Monogenia,  the 
eggs  develop  into  a  condition  suitable  for  the  invasion  of 
their  definitive  host  without  the  intervention  of  an  inter- 
mediate host,  and  there  is  no  alternation  of  generations  or 
asexual  multiplication.  From  one  egg,  therefore,  one 
sexually  mature  form  is  developed.  The  eggs  of  the  other 
and  more  important  division,  Digenia,  require  for  their 
development  an  intermediate  host,  and  there  is  alternation 
of  generations,  asexual  multiplication,  and  from  one  egg 
several  sexual  forms  may  ultimately  develop. 

The  full  life-history  of  the  human  Trematodes  is  not 
known.  Of  those  in  lower  animals  that  of  Fasciola  hepatica 
has  been  thoroughly  worked  out.  A  ciliated  embryo  is 
formed  in  the  egg  and  escapes,  the  miracidium.  It  then 
passes  into  a  fresh-water  snail.  In  the  liver  of  tlie  snail  it 
becomes  hollowed  out,  forming  a  sporocyst.  Buds  form 
in  the  interior  of  this  cyst  and  secondary  larvae,  redice,  are 
formed.  These  escape  into  the  tissues  of  the  snail,  and 
by  a  further  process  of  internal  budding  form  tertiary 
larvae  or  cercarice,  which  have  a  sucker,  and  escaping 
from  their  host  are  taken  up  with  grass  by  their  definitive 
host,  the  sheep.  They  pass  up  the  bile  ducts  into  the 
liver  of  this  animal  and  there  develop  into  the  sexually 
mature  form. 

In  other  species  the  development  of  the  redise  require  a 
second  intermediate  host. 

The  Trematodes  found  in  man  belong  to  four  families  : 

(i)  The  Monostomidce,  which  have  only  one  sucker,  are 
represented  by  the  Monostoma  lentis,  found  in  the  super- 
ficial layer  of  the  crystalline  lens  on  one  occasion  only. 

(2)  The  Fasciolidcv  have  two  suckers,  one  terminal  and 
the  other  ventral.  Of  this  family,  representatives  of  eight 
genera  are  found  in  man.  In  six  of  these  the  ventral 
sucker  is  near  the  oral  sucker.  Dicrocoslium,  in  which  the 
testicles  are  in  front  of  the  female  genital  organs;  Fasciola, 
Fasciolopsis,  Fasciolctta,  OpistliorcJiis  and  Clonorchis,  in  which 
the  testicles  are  behind  the  female  genital  organs,  and  in 
these  the  ventral  sucker  is  in  the  anterior  part  of  the  ventral 


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356  CLASSIFICATION    OK    FLUKES 

surface,  not  far  away  from  the  oral  sucker.  These  three 
genera  are  separated  from  each  other  by  the  shape  of  the 
anterior  extremity,  which  is  conical  in  the  Fasciola  ;  by 
the  character  of  the  intestinal  caeca,  which  are  much 
branched  in  Fasciola,  unbranched  but  sinuous  in  Fascio- 
lopsis,  and  unbranched  but  nearly  straight  in  Clonorchis. 
Fasciolopsis  and  Fascioletta  are  further  distinguished  by 
the  great  size  and  depth  of  the  acetabulum  or  ventral 
sucker,  and  by  the  conspicuous  and  long  cirrhus.  In 
Fasciola  and  Clonorchis,  as  well  as  in  Dicrocaelium,  the 
ventral  suckers  are  about  the  same  size  as  the  oral  suckers. 
In  the  other  two  genera  the  ventral  is  nearly  in  the  middle 
of  the  ventral  surface.  Heterophyes,  in  which  the  genital 
opening  is  behind  the  posterior  sucker,  which  is  very 
large,  much  larger  than  the  oral  sucker  ;  and  Paragonimus, 
in  which  the  genital  opening  may  be  median,  or  right  or 
left  of  the  middle  line.  The  ventral  sucker  is  about  the 
same  size  as  the  oral  sucker. 

(3)  The  Purampliistomirftv  have  two  suckers,  both  ter- 
minal, one  at  the  one  end  and  the  other  at  the  other  end  of 
the  animal.  They  include  two  divisions,  species  of  each  of 
which  are  parasitic  in  man — Amphistomum,  or  Paramphis- 
tomum,  and  Gastrodiscus.  These  genera  are  distinguished 
by  their  external  appearance,  as  in  the  Gastrodiscus  the 
anterior  extremity  is  conical  and  appears  to  rise  as  a 
projection  from  the  dorsal  surface  of  a  flat,  rounded  mas> 
which  contains  the  organs  of  reproduction.  The  human 
species  is  Gastrociiscits  hominis.  In  the  Amphistomum  or 
Paramphistomiim  the  shape  is  more  or  less  conical,  and 
there  is  no  marked  division  between  the  anterior  and 
posterior  part  of  the  body.  The  human  representative 
is  Amphistomum  watsoni.  This  is  more  correctly  a 
Cladorchis,  as  there  are  pharyngeal  pouches  which  are  not 
present  in  the  Paramphistoma.  In  both  Par  amphistomum 
and  Gastrodiscus  the  opening  of  the  genital  pore  is  in  the 
middle  of  the  body  and  not  near  the  acetabulum. 

(4)  Sc/iistosomiihv  have  two  suckers,  but  the  male  and 
female  organs  are  in  separate  animals. 


NEMATODES 


357 


Nematodes. 

The  commoner  Nematodes  found  in  the  human  in- 
testine are  the  Ascaris  lumbricoides,  rarely  Ascaris  mystax, 
Gnathostoma  siamense,  Oxyuris  vermicularis,  Ankylostomum 
duodenale,  Necator  americanus,  Strongylus  subiilis,  Tricho- 
cephalus  dispar,  Trichina  spiralis,  Strongyloides  intestinalis 
(A  nguillula  intestinale) . 

The  larger  worms  are  readily  found,  but  some  of  the 
smaller  ones  are  only  to  be  seen  by  very  careful  inspec- 
tion. The  Nematodes  are  liable  to  shrink,  rupture,  or 
otherwise  become  distorted,  and  are  not  very  easy  to 
stain.  As  a  general  rule  it  is  best  to  examine  them  un- 
stained, and  describe  them  as  they  appear  in  the  fresh 
condition,  mounted  in  normal  saline  solution.  For  per- 
manent specimens  fixation  in  70  per  cent,  alcohol  just  on 
the  boil  gives  good  results.  The  70  per  cent,  aqueous 
solution  of  alcohol  should  be  heated  till  bubbles  begin  to 
form,  and  the  living  worms  dropped  into  it.  The  lamp 
should  be  put  out,  and  the  worms  left  in  the  fluid.  After 
this  treatment  they  may  be  mounted  in  glycerine  jelly 
without  shrinking. 

Ascaris  lumbricoides. — These  are  large  round  worms. 
The  males  are  15  to  17  cm.  in  length  and  2  or  3  mm. 
in  breadth.  The  female  is  rather  larger,  20  to  25  cm. 
long  and  5*5  mm.  in  breadth.  These  worms  are  found 
in  any  part  of  the  intestinal  tract  and  occasionally 
pass  through  the  common  duct  into  the  gall-bladder 
or  even  the  biliary  ducts.  They  have  been  found  in 
hepatic  abscesses.  They  may  be  numerous  in  the  intestine. 
Rarely  Ascaris  mystax,  commonly  a  parasite  of  the  cat, 
a  smaller  ascaris,  with  lateral  alar  cuticular  appendages 
on  cephalic  end  of  the  body,  is  found  in  man. 

Oxyuris  vermicularis  is  a  small  cylindrical  worm  which 
tapers  towards  the  tail  to  a  sharp  point.  The  male  is  3  to 
5  mm.  in  length  and  at  the  tail  is  coiled  up  in  a  spiral. 
The  female  is  9  to  12  mm.  in  length.  This  worm  is  found 
in  the  whole  length  of  the  large  intestine  and  rectum  and 
may  escape  through  the  anus  (fig.  147,  a,  b).     The  males 


353 


ASCARIDES   AND   OXYURID.K 


are    found   higher  up   in  the  alimentary  canal  than   the 
females. 

Trichocephalus  disipar  (Whip-worm). — The  characteristic 
of  this  worm  is  a  long,  thin,  anterior  portion  somewhat 
resembling  the  lash  of  a  whip.  The  male  is  35  to  45  mm. 
in  length,  and  the  female  35  to  50  mm.  These  worms 
are  found  commonly  in  the  caecum  and  also  in  the 
ascending  and  transverse  colon.  They  are  very  rarely 
found  in  the  ileum  (fig.   142). 


FlG.  147.  —  Oxyuris  vermicularis.     a,  Male;   6,  female. 


Fig.  I4S. —  Trichocephalus  dispar.     a,  Male;  b,  female. 

The  Ankylostomum  duodenale  is  of  the  greatest  import- 
ance. These  worms  are  found  in  the  small  intestine 
and  may  be  very  numerous.  Both  males  and  females 
are  found.  They  fix  themselves  to  the  intestinal  wall 
and  live  on  epithelium,  debris,  &c,  but  not  blood.  The 
female  adult  worms  are  7  to  15  mm.  in  length  and  '8  mm. 
in  breadth.  They  have  a  mouth  surrounded  by  a  power- 
ful armature  consisting  of  two  pairs  of  curved  teeth  on  the 
posterior  wall  of  the  opening  and  of  two  triangular  plates 
terminating  in  sharp  points  anterior  to  the  mouth.     The 


Fig.    149.— m,f,  Male  and  female  Ankylostomes  ;  a,  head  of  A.  duodenale 
b,  head  of  Necator  americamts. 


360  ANKYLOSTOMUM    AND   NECATOR 

intestine  is  nearly  straight  and  commences  as  a  powerful 
oesophagus.  The  anus  is  subterminal  in  the  female.  The 
genital  opening  is  posterior  to  the  middle  of  the  body. 

The  males  are  rather  smaller  in  length,  6  to  11  mm., 
and  *5  mm  in  breadth;  they  have  similar  mouth-parts. 
The  caudal  extremity  is  expanded  into  a  membranous 
fold  of  the  integument  divided  into  four  unequal  lobes, 
of  which  the  lateral  ones  on  each  side  are  the  largest. 
There  are  two  equal  spicules  which  can  be  protruded 
through  the  cloaca  (fig.  149,  ;//). 

The  dorsal  lobe  is  of  importance,  as  it  differs  in  the 
character  of  the  supporting  rib  from  that  found  in  Necator 
americanus.  In  the  ankylostome  it  commences  as  a 
single  rib  which  only  bifurcates  in  the  peripheral  third 
and  finally  divides  into  three  minute  divisions  (fig.  150,  a). 

Necator  americanus  resembles  the  ankylostome  in  its 
habits  and  general  characters,  but  differs  in  that  the  two 
pairs  of  curved  teeth  are  replaced  by  sharp  chitinous 
thickenings,  and  that  in  the  female  the  genital  pore  is 
anterior  to  the  middle  line,  whilst  in  the  dorsal  flap  of 
the  caudal  bursa  of  the  male  the  supporting  rib  bifurcates 
near  the  base,  and  each  of  the  divisions  divides  into  two 
instead  of  into  three  as  in  the  ankylostome.  The  spicules 
in  Necator  americanus,  are  barbed  at  their  free  ends  like 
fish-hooks.  It  appears  to  be  the  indigenous  species  in 
Africa  and  in  some  parts  of  Asia. 

The  ankylostome  is  supposed  to  gain  access  to  the 
body  by  the  mouth,  but  it  has  been  shown  to  be  capable 
in  its  larval  form  of  penetrating  the  skin,  and  some 
experiments  seem  to  show  the  possibility  of  these  larval 
forms  obtaining  access  to  the  intestine  after  penetration 
of  the  skin  by  devious  routes.  In  some  cases  certainly 
passing  through  the  lungs  into  the  bronchi  and  then 
down  to  the  stomach. 

The  ankylostome  eggs  hatch  quickly,  within  forty- 
eight  hours,  and  the  embryos  rapidly  increase  in  size. 
If  kept  in  the  faeces  they  soon  die,  but  if  allowed  to  escape 
into  the  earth  they  undergo  further  development,  but  do 


STRONGYLOIDES    INTEST1NALIS 


36l 


not  become  sexually  mature,  or  reproduce  outside  the 
body.  In  their  final  stages  they  are  enclosed  in  their  old 
larval  skin  as  a  sheath  and  are  inactive  for  long  periods. 


Fig.  150. 


-a,  a,  Head  and  tail  of  male  A.  duodenale  :  b,  b,  head  and  tail 
of  male  N.  americanus. 


Strotigyloides  iutestinalis,  AnguiUula  intestinalis,  or 
Rhabdonema  intestlnale.  —  This  is  a  small  worm  only 
1  mm.  long  and  50  jul  in  breadth.     It  is  found  in  the  small 


362  TRICHINA    SPIRALIS 

intestine  and  the  maie  is  not  known.  Only  a  small 
number  of  eggs  are  formed,  four  or  five  as  a  rule.  The 
embryos  hatch  out  either  whilst  still  in  the  adult  or  when 
discharged  into  the  intestine.  These  embryos  can  be 
distinguished  from  Ankylostome  larvae  by  the  shape  of 
the  mouth  capsule.  The  embryos  outside  the  body 
are  capable  of  full  development  to  sexual  maturity 
and  reproduction.  These  free-living  sexual  forms  differ 
in  all  respects  from  the  parasitic  sexual  forms.  They  are 
shorter  and  broader,  the  oesophagus  has  a  double  dilata- 
tion and  the  embryos  are  more  numerous.  The  free- 
living  forms  must  alternate  with  a  parasitic  generation 
for  the  continued  existence  of  the  species. 

Trichina  spiralis. — The  adult  forms  are  found  only  in 
the  intestines  and  intestinal  walls  of  man,  pigs,  rats,  &c. 
They  are  found  only  for  a  period  of  a  few  weeks  after 
eating  flesh  in  which  encysted  embryos  were  present. 
The  cysts  are  dissolved  in  the  stomach,  the  embryos  are 
set  free,  and  in  the  walls  of  the  human  small  intestine 
pass  through  several  metamorphoses  and  become  sexually 
mature  in  a  few  days. 

The  mature  worms  are  just  visible  to  the  naked  eye. 
The  male  measures  1  to  5  mm.  in  length  and  "04  mm.  in 
breadth.  There  is  a  straight  intestine  with  a  powerful 
oesophagus.  The  anus  opens  into  a  terminal  cloaca  into 
which  also  the  vas  deferens  from  the  single  testicular 
xube  opens.  There  are  two  digitiform  appendages,  one 
on  each  side  of  the  cloaca,  which  serve  as  copulatory 
organs.     There  are  no  spicules. 

The  female  is  larger,  3  to  4  mm.  in  length  and  *o6  mm. 
in  breadth.  There  is  a  single  ovarian  tube,  which  is 
continued  into  a  widely  dilated  portion,  the  uterus,  from 
which  a  narrow  tube  leads  to  the  genital  opening,  which 
is  situated  about  the  junction  of  the  anterior  fourth  with 
the  rest  of  the  body. 

The  embryos  are  passed  alive,  but  do  not  appear  in 
the  faeces,  as  they  pass  through  the  intestinal  wall,  and 
finally  reach  the  muscles  and  there  become  encysted. 
They  may  become  encysted  in  the  intestinal  walls. 


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364  PROTOZOA 

Man  can  therefore  be  both  the  intermediate  and 
definitive  host,  but  many  other  animals  harbour  the 
parasites,  particularly  pigs  and  rats.  It  is  from  badly 
cooked  pork  that  man  usually  becomes  infected.  Pigs 
are  probably  more  frequently  infected  from  rats. 

To  find  the  adults  the  intestinal  contents  of  the  upper 
part  of  the  small  intestine  should  be  examined  with  a 
low  power.  The  worms  may  be  found  by  examination 
with  a  simple  lens,  but  are  easily  overlooked.  The 
mucosa  must  also  be  scraped  off  and  examined,  as  the 
worms  soon  penetrate  into  it.  The  encysted  embryos 
are  seen  as  white  specks  in  the  muscles,  and  are  most 
numerous  towards  the  insertion  of  the  muscle.  They 
may  be  found  in  fat  and  other  tissues,  but  are  less  easily 
seen  in  such  situations. 

Examination  of  the  intestinal  contents  for  small  worms 
is  facilitated  by  dilution  of  the  stool  with  normal  saline 
solution.  The  mixture  can  be  well  shaken,  and  the 
worms  if  viewed  against  a  black  background  are  more 
readily  seen.  With  small  animals  the  unopened  intes- 
tines may  be  cut  into  lengths  of  an  inch  or  two,  and  by 
scraping  on  the  flattened  intestine  in  one  direction  the 
contents  and  parts  of  the  mucous  membrane  are  pressed 
out  of  the  intestine,  and  in  this  expressed  substance 
worms  may  be  found.  The  portion  of  the  intestine  can 
then  be  slit  open  and  placed  between  two  slides  and 
examined  for  encysted  worms.  If  too  opaque  it  may 
be  placed  in  weak  sodium  hydrate  solution,  which  will 
in  a  minute  or  two  render  it  transparent,  and  the  worms 
will  be  more  readily  seen. 

Protozoa. 

Protozoa  belonging  to  various  orders  are  found  in  the 
stools.  Of  these  the  Amoeba  coli,  a  large  amoeba  which 
is  passed  with  the  mucus  and  in  the  faeces  in  many 
chronic  and  recurrent  dysenteries,  is  of  the  most  import- 
ance. Some  observers  state  that  it  is  found  in  normal 
stools,  and  it  is  certainly  found  in  stools  of  patients  who 


AMCEBA  365 

do  not  complain  of  either  diarrhoea  or  dysentery-  In  a 
large  proportion  of  these  cases  it  will  be  found  that 
mucus  is  passed  with  each  stool,  and  in  some  ulceration 
of  the  colon  has  been  present  and  found  on  f>ost-mortew 
examination.  In  other  cases  amoebae  are  found  in  persons 
apparently  healthy. 

In  a  fresh  stool  the  diagnosis  is  easy,  particularly  in  a 
warm  tropical  country,  or  where  a  hot  stage  is  used.  The 
large  cells  with  active  amoeboid  movement  often  contain- 
ing in  their  interior  red  corpuscles  and  food  vacuoles, 
permit  of  no  mistake.  If  the  stool  has  been  some  time 
passed,  allowed  to  cool  or  treated  with  antiseptics,  dia- 
gnosis is  less  easy,  as  the  amoebae,  when  they  die,  become 
globular,  and  are  not  then  easily  distinguished  from 
other  large  cells  in  the  mucus.  If  they  contain  blood 
corpuscles  or  other  substances  taken  as  food,  they  can  be 
more  readily  recognized.  They  stain  well  with  any  basic 
stain,  but  there  is  no  satisfactory  differential  stain.  For 
the  study  of  stained  specimens  smears  of  the  faeces  or 
mucus  should  be  fixed  whilst  still  wet,  by  placing  them  in 
a  saturated  solution  of  perchloride  of  mercury  for  ten 
minutes.  They  are  then  well  washed  in  running  water, 
and  can  be  stained  with  borax  carmine,  or  by  the  iron 
alum  haematoxylin  (see  Appendix). 

The  life-history  of  the  different  amoebae  is  not  fully 
confirmed.  In  the  commoner  intestinal  amoebae  asexual 
multiplication  is  by  simple  fission.  This  occurs  in  the 
intestine  and  in  hepatic  abscesses.  Under  certain  con- 
ditions an  amoeba  will  become  encysted,  and  the  contents 
then  divide  after  a  series  of  changes  partly  outside  the 
body  into  eight  young  amoebae.  These  encysted  forms 
are  resistant,  and  retain  their  vitality  for  a  long  time 
outside  the  body.  They  are  probably  the  important 
agents  in  the  propagation  of  the  parasite.  The  patho- 
genic properties  are  disputed  by  some.  In  the  most 
severe  cases  of  dysentery  the  Amoeba  coli  is  not  found, 
and  it  is  usually  absent  in  epidemic  dysentery.  It  is 
most  frequently  found  in  relapsing  or  recurrent  dysentery. 

Amoebae   are   found    in   the  pus  of  hepatic  abscesses. 


$66 


AMCEBA 


They  are  very  difficult  to  find  in  the  pus  discharged  at 
first.  If  the  pus  be  examined  three  or  four  days  after  the 
abscess  is  opened  they  are  usually  readily  found. 


Asexual  multiplication  by  simple  division  of  nucleus  and  cytoplasm. 


Fig.  151. — Scheme  of  Development  of  Amneba.  Multiplication  in  encysted 
forms  (autogamous).  ?  sexual  multiplication.  The  early  stages  of  division 
of  the  nucleus  (a — d)  and  conjugation  of  the  divided  nuclei  in  pairs  (e), 
followed  by  further  division  of  these  products  of  conjugation,  first  into  two 
and  then  into  four  each  (f—  A).  The  thick  wall  of  the  cyst  in  the  later  stages 
indicates  the  hardening  of  the  cyst  wall  during  the  stages  when  the  cysts  are 
outside  the  body.  Stages  a — h  take  place  within  the  body.  Stage  i  is 
only  found  outside  the  body,  whilst  stage  /'  is  believed  to  occur  in  the  stomach 
of  a  second  host. 


Schaudinn  differentiates  two  distinct  species  of  Amoeba 
in  the  human  intestine.  The  one,  the  Entamoeba  coli,  may 
be  present  in  the  intestines  of  healthy  persons,  and  does 
not  invade  the  tissues  or  pass  into  other  organs  in  the 
body.     The  other,  which  he  named  Entamoeba  histolytica, 


ENTAMOEBA   HISTOLYTICA  367 

does  invade  the  tissues  of  the  alimentary  canal,  and 
may  pass  to  various  parts  of  the  body,  liver,  &c.  The 
morphological  differences  in  the  amoeba  as  seen  in  the 
stool  are  slight.  In  the  Entamoeba  coll  the  ectosarc  is  not 
clearly  differentiated  from  the  endosarc  when  the  amoeba 
is  at  rest.  The  pseudopodia  are  formed  from  both 
ectosarc  and  endosarc.  The  nucleus  is  large  and  rich 
in  chromatin.  Development  is  as  described  for  the  type 
in  main  essentials. 

Entamoeba  histolytica  has  a  small  nucleus  poor  in 
chromatin  and  placed  eccentrically.  In  the  resting 
amoeba  the  ectosarc  is  clearly  denned  and  pseudopodia 
are  formed  from  the  ectosarc  only.  The  development 
also  differs.  Multiplication  may  take  place  (1)  by  simple 
fission,  as  in  Entamoeba  coll ;  (2)  by  irregular  gemmation, 
the  number  of  young  amoebae  formed  being  indefinite  ; 
or  (3)  autogamy,  in  which  the  nucleus  breaks  up  into 
a  number  of  chromidia  which  are  diffused  through  the 
protoplasm.  Secondary  nuclei  then  form  at  the  periphery 
from  which  spores  are  produced.  These  are  surrounded 
by  a  yellowish  cyst  wall  and  are  the  resistant  forms. 

In  this  amoeba,  therefore,  the  resistant  encysted  forms 
are  young  spores,  whilst  in  Entamoeba  coll  it  is  the  mature 
form  which  is  encysted  and  the  spores  are  formed  inside 
the  cyst.  Others  view  with  suspicion  all  amoebae  and 
consider  that  some  of  the  free-living  forms  may  become 
parasitic  and  pathogenic. 

Coccidia  are  said  to  have  been  found  in  human  faeces. 

Various  flagellated  organisms  have  been  described  in 
the  stools.  One  is  Cercomonas  liomlnls.  It  is  a  small 
round  body  with  one  or  two  long  flagella.  It  is  rarely 
found  in  healthy  stools  but  may  be  common  in  some 
cases  of  diarrhoea.  Recently  doubt  has  been  cast  on  the 
existence  of  Cercomonas. 

Other  intestinal  flagellates  which  merit  attention  are 
Trichomonas  and  Lamblia. 

Trichomonas  (fig.  152)  is  pyriform  in  shape,  the  anterior 
end  being  rounded,  whilst  the  posterior  is  pointed.     At 


368 


CERCOMONAS 


the  anterior  end  there  are  three  flagellar  of  equal  length 
and  fused  together  at  their  base.  There  is  also  an  un- 
dulating membrane  arising  at  the  anterior  extremity  and 
running  obliquely  backwards.  The  nucleus  is  situated 
near  the  anterior  extremity.  This  parasite  seems  to  be 
quite  harmless  to  its  host. 


(After  Wenyon). 

Fig.  152. — A,  Lamblia ;  n,  nucleus;  /.  flagellum.  b,  Lamblia,  side  view  ; 
n,  nucleus  ;  /,  flagellum.  c,  Trichomonas  ;  n,  nucleus  ;  f,  flagellum  ; 
m,  undulating  membrane. 


Lamblia  (fig.  152)  occurs  usually  in  the  duodenum  or 
jejunum.  It  has  a  pear-shaped  flattened  body,  with  a 
large  sucker-like  depression  on  the  ventral  surface,  by 
which  it  adheres  to  epithelial  cells.  It  has  four  pairs 
of  flagella,  all  directed  backwards,  and  a  double  nucleus. 


INFUSORIA 


3^9 


This  parasite  is  probably  pathogenic.  The  symptoms  are 
of  a  chronic,  recurrent  diarrhoea,  with  abundant  dis- 
charge of  mucus,  often  bile-stained  and  frequently  mixed 
with  the  faeces  and  sometimes  with  blood. 

When  there  is  diarrhoea  the  parasite  may  be  found  in 
abundance  ;  at  other  times  only  encysted  forms,  devoid 
of  any  flagella,  will  be  found. 

Flagellated  organisms  have  also  been  found  in  the 
mouth  and  in  abscesses  in  connection  with  the  mouth 
cavity. 


Fig.  153. 


Spirochaetae  are  sometimes  found  in  healthy  stools,  and 
are  common  in  some  cases  of  dysentery. 

Infusoria  are  found  in  some  cases  of  diarrhoea  ;  the 
best  known  resemble  a  large  Paramcecium — Balantidium 
coll.  It  measures  65  to  85  /x  in  length.  It  may  be  found 
in  very  large  numbers  in  the  stools,  and  in  such  cases 
it  may  also  be  found  in  the  intestinal  walls  and  even  in 
the  blood-vessels  ;  it  has  been  found  in  the  pus  of  an 
abscess  of  the  liver.  It  is  probably  pathogenic.  It  is  a 
24 


3/0  VEGETABLE   MICRO-ORGAN  ISMS 

parasite  found  very  commonly  in  the  intestines  of  pigs 
(fig-  I55)-  It  differs  from  Paramcecium  in  the  characters 
and  arrangement  of  the  cilia  guarding  the  peristome. 

Vegetable  micro-organisms  abound.  Many  of  these 
belong  to  the  colt  group,  and  include  organisms  which 
are  harmless  and  others  which  are  pathogenic.  Many 
of  the  organisms,  as,  for  instance,  the  Bacillus  coli  com- 
munis, though  harmless  to  persons  in  good  health  as  long 
as  they  are  contained  in  the  alimentary  canal,  can,  under 
certain  circumstances,  invade  the  tissues  and  then  become 
actively  pathogenic  and  in  some  cases  pyogenic.  The 
intestinal  mucosa  possesses  considerable  power  of  resist- 
ance even  to  many  decidedly  pathogenic  organisms,  and 
consequently  attempts  at  infection  by  the  imbibition  of 
cultures,  &c,  often  fail. 

Impaired  resistance  due  to  bad  health,  malnutrition, 
combined  with  enhanced  virulence  of  an  organism,  is 
necessary  in  many  cases  for  even  pathogenic  organisms 
to  cause  disease. 

The  isolation  and  identification  of  pathogenic  and  non- 
pathogenic organisms  in  the  alimentary  canal  is  a  matter 
of  considerable  difficulty  and  complexity  on  account  of 
the  large  number  and  species  of  organisms  normally 
present. 


371 


CHAPTER  XXI. 

Urine. 

It  is  not  proposed  to  consider  the  ordinary  tests 
for  the  abnormal  constituents  of  urine,  such  as  albumin, 
sugar,  and  the  like,  but  only  a  few  special  points  in  con- 
nection with  the  examination  of  urine  in  the  Tropics. 

Blood  is  found  under  special  circumstances  as  a  result 
of  parasitic  invasions  by  Filaria  nocturna  and  ScJiisto- 
somum  haematobium  (bilharzia)  respectively,  and  there  is 
at  least  one  form  of  tropical  haemoglobinuria — black- 
water  fever. 

Hematuria  can  be  easily  distinguished  from  haemo- 
globinuria by  the  presence  of  red  corpuscles  in  the  deposit. 
In  many  cases  it  is  easily  distinguished  with  the  naked 
eye,  as  the  superjacent  fluid  may  not  be  coloured  with 
blood  in  haematuria.  If  coloured  with  blood  it  is  cloudy 
•and  not  a  clear  and  transparent  red,  as  is  the  solution  of 
haemoglobin  in  haemoglobinuria. 

In  haematuria  from  bilharzia  infection  the  bulk  of  the 
urine  is  often  free  from  blood,  but  if  the  patient,  after 
apparently  emptying  the  bladder,  strains,  the  last  few 
drops  may  be  found  to  contain  blood  or  mucus,  and  in 
this  blood  or  mucus  the  characteristic  ovum  with  its 
terminal  spike  and  the  contained  ciliated  embryo  will  be 
found.  In  all  suspected  cases  it  is  therefore  necessary  for 
the  patient  to  strain,  and  the  few  drops  so  passed  are  the 
most  important  for  examination. 

Schistosoma  have  been  found  not  only  throughout 
Africa,  in  Arabia  and  Cyprus,  but  also  in  some  of  the 
smaller  West  Indian  Islands,  and  it  is  therefore  possible 
that  bilharziosis  may  become  a  more  widely  diffused 
disease  than  is  at  present  the  case. 


."»/  * 


ILK MAI  TK1A 


In  some  of  the  places,  as  in  the  West  Indies,  it  is  reported 
that  the  Schistosoma  eggs  are  found  only  in  the  faeces, 
and  that  hematuria  does  not  occur.  It  is  believed  by 
some  that  the  eggs  passed  f>er  rectum  belong  to  a  different 
undescribed  Schistosomum  which  has  been  named  S. 
mansoni.  The  eggs  of  Schistosomum  japonicum  are  not 
found  in  the  urine  nor  is  haematuria  caused  by  them. 

In  most  cases  of  bilharziosis  there  will  be  a  history  of 
occasional  attacks  of  haematuria.  In  these  cases,  by 
finding  the  ova  in  the  last  few  drops  of  urine  expressed 
from  the  bladder,  the  causation  of  the  disease  can  be 
determined. 

Haematuria  from  filariasis  is  rarely  an  admixture  of 
blood  only.  More  often  chyle  is  also  present  and 
usually  chyle  occurs  without  any  admixture  with  blood 
(chylnria).  Coagulation  of  the  chyle  and  blood  fre- 
quently takes  place,  so  that  clots  of  blood-stained  sub- 
stance, or  of  pure  blood,  are  formed,  or  the  whole  mass 
may  set  as  a  pinkish  jelly. 

The  embryos  of  the  Filaria  bancrofti  may  be  found 
in  the  urine,  but  are  more  abundant  in  the  blood.  If 
scanty  any  small  masses  of  blood  or  filaments  of  thread 
should  be  examined  as  the  filariae  often  adhere  to  them. 
Some  authors  advise  filtering  the  urine,  and  in  the  last 
few  drops  left  in  the  filter  the  embryos  will  be  found. 
There  is  no  periodicity  in  the  appearance  of  the  filarial 
embryos  in  the  urine.  If  filariae  are  not  found  in  the 
urine  they  may  be  found  in  the  peripheral  blood  of  the 
patient  if  the  examination  be  made  at  night. 

Haematuria  may  also  result  from  other  causes,  such  as 
calculus,  malignant  disease,  &c,  but  those  are  not  limited 
to  the  Tropics. 

Hemoglobinuria,  or  the  passage- of  urine  coloured  with 
dissolved  haemoglobin,  is  the  characteristic  of  "black- 
water  fever."  Cases  of  paroxysmal  haemoglobin uria 
would,  no  doubt,  if  they  occurred  in  an  endemic  area, 
be  mistaken  for  blackwater  fever.  Haemoglobinuria  is 
met  with  in  Africa  as  a  common  disease,  in  some  places 
10  per  cent,  of  the  most  susceptible  portion  of  the  popu- 


HEMOGLOBINURIA  373 

lation  (European)  are  attacked  annually,  or  one-quarter 
of  that  proportion  of  the  less  susceptible  Asiatics.  In 
some  parts  of  India  a  fair  number  of  cases  are  met  with, 
but  only  in  small  proportions  as  compared  with  Africa. 
Cases  are  reported  from  other  malarial  countries,  South 
America,  West  Indies,  Soloman  Islands,  Malaya,  South 
of  Europe,  &c,  but  the  disease  is  less  common  in  those 
countries. 

The  urine  when  first  passed  is  clear  and,  when  diluted 
sufficiently,  is  transparent,  any  deposit  present  is  mainly 
of  casts  and  epithelium.  As  it  cools,  and  particularly 
when  it  becomes  alkaline,  a  thick  amorphous  albu- 
minoid and  brown  deposit  is  thrown  down.  The  greater 
the  dilution  required  to  render  the  urine  transparent  the 
more  concentrated  is  the  haemoglobin  solution  in  the 
urine,  and  the  larger  the  amount  of  the  haemoglobin  the 
more  severe  and  protracted  will  be  the  attack. 

Rate  of  Secretion.— -In  this  and  also  in  yellow  fever  the 
rate  of  secretion  of  the  urine  is  a  matter  of  great  import- 
ance, as  if  the  urine  is  much  diminished  the  prognosis 
is  grave  and  active  measures  are  urgently  required.  The 
times  of  micturition  and  the  amount  passed  each  time 
must  be  observed,  and  the  amount  of  urine  passed  at 
each  micturition,  divided  by  the  number  of  hours  that 
have  elapsed  since  the  previous  micturition,  will  give  the 
rate  of  excretion  per  hour  during  that  period.  ^Any  fall  in 
this  rate  is  an  important  warning.  If  suppression  is  once 
established  recovery  will  not  take  place  in  either  disease. 

Bile  in  the  urine  may  occur  in  some  cases  of  malaria 
as  a  transient  phenomenon.  The  persistent  presence  of 
bile  in  an  acute  attack  of  malaria  is  a  rare  but  a  serious 
and  frequently  fatal  complication. 

Bile  is  met  with  in  the  urine,  frequently  in  relapsing 
fever,  and  is  not  very  rare  in  lobar  pneumonia. 

There  are  cases  of  jaundice  occurring  in  the  Tropics, 
associated  with  high  fever,  which  are  neither  yellow  fever 
nor  malaria.  These  require  investigation.  Nothing  is 
known  of  the  true  nature  of  these  diseases.  Possibly 
some  of  them  are  cases  of  Weil's  disease. 


374  INDICAN 

Haemoglobinuric  urine  can  be  distinguished  from 
bilious  urine  by  dilution,  when  the  red  colour  of  the 
haemoglobin  is  seen.  By  shaking  the  urine  and  noting 
the  pink  tinge  of  the  froth  as  compared  to  the  yellow 
tinge  of  the  froth  of  bilious  urine,  the  distinction  is 
readily  made. 

The  most  satisfactory  method  for  diagnostic  purposes 
is  the  use  of  a  spectroscope,  when  the  haemoglobin  bands 
will  be  clearly  seen  (vide  Table  of  Spectra).  In  some  of 
the  cases  all  through,  and  in  others  at  onset  and  end  of 
an  attack,  methaemoglobin  is  passed  alone.  Such  urine 
is  of  a  brownish  colour  and  can  only  be  distinguished  by 
the  spectroscope  (spectra  4  and  5).  There  is  reason  to 
believe  that  many  mild  cases  of  blackwater  fever  are 
overlooked,  as  the  urine  contains  only  this  methaemo- 
globin. In  this  disease  casts  are  present  often  in 
large  numbers ;  the  casts  are  granular,  do  not  often 
include  epithelial  cells,  but  generally  contain  granules 
of  bright  yellow  pigment  derived  from  the  haemo- 
globin. Such  casts  are  found  for  weeks  after  an  atlack 
of  blackwater  fever,  though  the  urine  is  free  from 
albumin. 

It  is  important  to  be  able,  in  watching  a  case,  to  form 
an  estimate  of  the  variations  in  the  amount  of  haemo- 
globin present.  This  is  readily  done  if  the  first  urine  be 
diluted  in  the  test  tube  to  a  convenient  known  extent. 
This  is  the  standard,  and  the  other  urines  found  are 
similarly  diluted  if  necessary  till  they  match  the  standard. 

Indican  is  very  commonly  present  in  the  urine  of 
patients  in  the  Tropics,  usually  in  cases  of  intestinal 
disorder.  It  is  best  detected  by  conversion  into  indigo 
blue.  The  simplest  method  is  to  place  a  crystal  of 
potassium  chlorate  at  the  bottom  of  a  tube  and  cover 
this  crystal  with  the  urine.  Strong  hydrochloric  acid 
is  allowed  to  run  down  to  the  crystal  without  mixing 
with  the  urine.  A  blue  ring  forms  at  the  point  of 
junction  of  the  two  fluids  if  indican  be  present. 

Nitric  Acid  Test. — Strong  nitric  acid  added  so  as  to 
form  a  layer  below  the  urine  will  lead  to  the  formation 


BACTERIA  375 

of  a  red  colour  at  the  junction  of  the  fluids.  If  little 
indican  is  present  this  colour  will  appear  in  five  to 
ten  minutes  ;  if  in  considerable  amount  it  will  appear 
at  once,  and  if  greatly  in  excess  will  be  almost  black. 

Bacteria  are  frequently  met  with  in  the  urine.  Of  the 
pathogenic  organisms,  the  warnings  which  will  be  given 
as  to  the  danger  of  confusing  the  smegma  bacillus  with 
tubercle  must  be  borne  in  mind.  The  typhoid  bacillus 
may  be  found  in  the  urine  for  prolonged  periods  after 
recovery  from  the  disease  ;  and  so  may  the  micrococcus 
of  Malta  fever.  In  systemic  infections  with  B.  coli 
communis  the  organism  is  frequently  present  in  the  urine. 
In  examinations  for  such  organisms  it  is  important  that 
the  urine  should  be  drawn  off  by  a  sterilized  catheter  and 
received  into  a  sterilized  vessel. 

Flakes  of  pus  or  muco-pus  require  careful  bacterio- 
logical examination.  Often  they  are  the  remnants  of  a 
gonorrhceal  infection  and  the  gonococcus  may  be  found 
abundantly.  Sometimes  they  are  due  to  tuberculosis. 
In  all  such  examinations  the  urine  first  passed  in  the 
morning  should  be  examined,  as  such  discharges  accu- 
mulate during  the  night.  The  urine  should  be  divided 
into  three  parts  :  that  first  passed  contains  the  washings 
of  the  urethra.  The  great  mass  of  the  urine  which  will 
contain,  with  little  admixture,  substances  derived  from 
the  kidneys  and  ureters ;  and  lastly  that  passed  after 
forcible  expression  of  the  last  few  drops,  which  will 
contain  discharges  from  the  wall  of  the  bladder  and  from 
prostatic  crypts. 

The  bacteriology  of  the  urine  in  the  Tropics  has 
received  little  attention,  but  there  are  many  cases  of 
systemic  infection  with  B.  coli  communis  which  originate 
in  the  Tropics,  and  in  which  the  organisms  are  found  in 
the  urine,  and  probably  there  are  cases  in  which  a  similar 
infection  with  other  organisms  takes  place. 

It  is  well  to  remember  that  urine  can  be  used  as  a 
medium  for  the  growth  of  organisms.  That  passed  about 
two  hours  after  a  meal  is  the  best,  as  it  is  not  too  acid. 


376  HEWLETT'S   BODIES 

B.  typhosus  and  many  others  grow  fairly  well.     It  requires 

boiling,  filtering  and  sterilization,  and  can  be  used  either 
as  a  fluid  medium,  or  by  the  addition  of  gelatine  or 
agar  made  into  a  solid  medium. 

Some  of  the  diseases  in  the  Tropics  are  attended  with 
deviations  from  normal  in  the  metabolic  and  catabolic 
processes  indicated  to  some  extent  by  changes  in  the 
urine.  The  diminution  in  the  amount  of  urea  in  beri- 
beri cases  is  an  instance  in  point. 

Other  changes  have  been  noticed  in  the  urine  of  beri- 
beri patients  and  deserve  close  investigation.  Hewlett 
has  noted  that  casts,  usually  hyaline,  but  sometimes 
granular,  can  always  be  found  in  centrifugalized  urine, 
and  in  addition  that  peculiar  refractile  bodies  are  present. 
These  he  describes  as  of  three  classes  :  (1)  Small  forms 
one-third  to  one-half  the  diameter  of  a  red  corpuscle, 
very  refractile  and  apparently  having  a  thick  capsule  with 
hyaline  contents ;  (2)  larger  forms,  12  to  20  /^,  spherical  in 
shape,  and  containing  refractile  granules,  and  appearing  to 
contain  also  what  may  or  may  not  be  a  nuclear  body; 
(3)  occasional  very  large  bodies,  30  /jl  in  diameter,  contain- 
ing fine  granules  and  a  distinct  nucleus  with  nucleolus. 

These  cannot  be  dried,  fixed  and  stained,  but  in 
normal  saline,  in  hanging  drop  preparations,  they  stain 
with  neutral  red,  methylene  blue  and  methyl  green,  but 
much  less  deeply  than  epithelial  cells  or  leucocytes. 

None  of  these  bodies  stain  with  osmic  acid  or  with 
Sudan  III.,  and  therefore  they  cannot  be  fat.  They  may 
be  myelin  bodies.  They  are  not  found  in  normal  urine 
or  in  that  of  persons  with  nephritis. 

Hewlett  suggests  that  they  may  be  the  result  of 
peculiar  degenerative  changes  in  cells,  or  possibly  are 
due  to  protozoa. 

Diazo-reaction  is  a  reaction  which  is  constant  in 
typhoid  fever.  The  reaction  may  occur  in  other  diseases, 
notably  miliary  tuberculosis,  measles,  scarlet  fever  and 
erysipelas,  usually  in  severe  cases  of  these  diseases.  The 
reaction  is  usually  to  be  obtained  about  the  fourth  to 
seventh  day  in  enteric  fever,  and  though  not  conclusive  as 


DIAZO-RKACTION  377 

a  test  as  it  occurs  in  other  diseases,  it  is  an  important  aid  in 
the  exclusion  of  enteric  in  obscure  cases  of  continued  fever. 
Two  solutions  are  required  for  the  test  : — 
Solution   1. 

Sulphanilic  acid  ...         ...  ...  2  grm. 

Hydrochloric  acid  ...  ...       50  c.c. 

Distilled  water      ...  ...  ...   1,000  c.c. 

Solution  2. 

Sodium  nitrite     ...  ...  ...        0*5  grm. 

Distilled  water     ...         ...         ...       100  c.c. 

One  part  of  solution  No.  2  is  added  to  fifty  parts  of 
solution  No.  1,  and  mixed  with  an  equal  quantity  of  urine 
in  a  test  tube.  This  mixture  is  then  rendered  strongly 
alkaline  by  ammonia. 

If  the  reaction  is  positive,  the  mixture  becomes 
carmine-red  in  colour,  and  if  the  test  tube  is  shaken,  this 
colour  is  seen  in  the  foam.  If  the  colour  does  not  appear 
in  the  foam,  the  reaction  is  negative. 

Estimation  of  Quinine  excreted  in  the  Urine. — It  is  occa- 
sionally necessary  in  treating  patients  with  quinine  to 
estimate  the  amount  of  that  drug  excreted  by  the  urine, 
and  for  that  purpose  the  method  recommended  by 
Christophers  and  Stephens  may  be  employed. 

Two  hundred  cubic  centimetres  of  urine  are  acidified 
with  a  few  drops  of  sulphuric  acid.  A  spoonful  of  solid 
picric  acid  is  then  added.  The  solution  is  allowed  to 
stand  for  an  hour,  and  then  filtered.  The  filtrate  should 
be  quite  clear,  and  should  give  with  a  saturated  solution 
of  picric  acid  no  turbidity.  If  there  is  difficulty  in 
getting  a  clear  filtrate,  add  a  trace  of  egg  albumin,  and 
again  filter.  The  residue  is  now  digested  in  an  Ehrlen- 
meyer  flask  with  50  cc.  of  3  per  cent,  soda  solution  for 
half  an  hour  on  the  water  bath.  Now  add  60  cc.  of 
chloroform,  and  shake  for  two  hours  in  a  shaking 
machine.  The  solution  of  chloroform  is  now  removed 
by  means  of  a  separating  funnel,  and  collected  in  a 
weighed  flask.  The  flask  should  have  a  long  neck  to 
prevent  spurting.  Evaporate  on  a  water  bath,  and  dry  at 
1200  C.     The  residue  is  quinine. 


37» 


CHAPTER  XXII. 
Bacteriology. 

The  pathogenic  micro-organisms  with  vegetable  char- 
acteristics are  less  generally  studied  in  the  Tropics  than 
elsewhere.  Much  of  the  easier  work  could  be  done 
without  complicated  apparatus  or  any  great  difficulty. 

The  methods  now  employed  in  British  laboratories 
require  too  much  apparatus,  and  are  too  complicated  to 
be  used  by  a  private  worker  in  the  Tropics,  and  only  the 
simpler  methods  which  are  at  his  disposal  are  considered 
here.  This  account  of  the  methods  which  can  be  used 
is  therefore  intended  only  for  those  obliged  to  use  primi- 
tive methods  and  makeshifts. 

For  the  isolation  and  cultivation  of  vegetable  micro- 
organisms artificial  media  are  necessary,  and  the  basis 
of  the  standard  media  is  nutrient  broth.  There  is  much 
difficulty  attending  the  making  of  nutrient  broth  from 
meat  in  the  Tropics,  but  meat  extracts,  particularly 
Bovril  or  Liebig's,  make  an  efficient  substitute.  In 
broth  prepared  from  either  of  these,  the  organisms  that 
will  grow  in  nutrient  broth  made  from  meat  will  grow 
fairly  well. 

An  iron  enamelled  jug,  measures,  scales,  and  weights, 
a  glass  rod,  a  funnel,  and  ordinary  filter  paper  or  white 
blotting  paper  is  all  the  apparatus  required.  Bovril 
peptone  and  common  salt  and  water  are  the  substances 
needed,  and  litmus  paper,  or  better,  phenolphthalein, 
which  is  required  for  the  neutralization  of  the  broth 
when  made,  as  well  as  a  carbonate  of  soda  or  sodium 
hydrate  solution. 

Nutrient    Broth. — To    make    the    broth  :    Take    1,000 


NUTRIENT   BROTH  379 

c.c.  or  i  litre  of  water  :  then  take  5  grm.  each  of  Bovril 
(or  Liebig)  and  salt,  and  10  grm.  of  peptone  (Wittes'  is 
usually  used).  Mix  the  peptone  with  about  25  c.c. 
of  the  water,  and  stir  it  well  so  as  to  form  a  kind 
of  emulsion  ;  then  to  this  add  the  remainder  of  the 
water  and  the  salt  and  Bovril.  The  Bovril  can  be 
conveniently  weighed  in  a  watch-glass,  or  if  Liebig  is 
preferred,  this  can  be  spread  with  a  spatula  on  a  piece 
of  filter  paper,  and  the  watch-glass  with  the  Bovril  in 
it,  or  the  filter  paper  with  the  Liebig's  Extract  on  it, 
can  be  placed  in  the  water  with  the  other  ingredients. 
The  whole  should  now  be  boiled  for  a  quarter  of  an 
hour,  and  well  stirred  to  ensure  thorough  solution.  It 
is  now  ready  for  neutralization.  When  made  with 
Liebig,  the  broth  will  be  much  too  acid  to  get  good 
growths,  and  with  Bovril,  though  much  less  acid,  may  be 
too  acid  to  be  quite  satisfactory.  Moreover,  the  degree 
of  acidity  of  different  specimens  varies. 

Neutralization. — Litmus  paper  can  be  used  in  an 
emergency  to  determine  the  reaction  of  the  broth,  but 
is  unsatisfactory,  as  many  of  the  organic  acids  do  not 
affect  litmus  paper,  and  the  dibasic  sodium  phosphates 
act  on  litmus  paper  as  an  alkali.  Many  specimens  of 
broth  also  have  a  double  reaction,  turning  red  litmus 
paper  blue  and  blue  litmus  paper  red,  so  as  to  leave 
the  point  of  neutralization  uncertain. 

Where  possible  phenolphthalein  should  be  used.  A  '5 
per  cent,  solution  of  phenolphthalein  in  spirit  is  the 
indicator.  This  solution  is  colourless  when  acid  or 
neutral,  but  turns  a  deep  magenta  colour  with  any  free 
alkali. 

Carbonic  acid  should  be  expelled  from  a  measured 
quantity  of  the  broth,  say  25  c.c.  by  boiling;  to  this  broth 
a  few  drops  of  the  phenolphthalein  should  be  added,  and 
then  drop  by  drop  the  alkaline  solution,  till  the  broth 
turns  a  flesh  or  faint  pink  colour,  indicating  that  the 
acid  is  completely  neutralized.  The  amount  of  alkaline 
solution  has  been  measured,  and  as  there  are  975  c.c.  of 


3*>o  NEUTRALIZATION 

broth  left  the  amount  required  for  the  neutralization 
of  the  25  c.c.  multiplied  by  '■'J-,5  =  39  will  give  the  amount 
of  the  alkaline  solution  required  for  the  neutralization  of 
the  remainder  of  the  broth. 

It  is  to  be  noted  that  to  exactly  neutralize  the  broth 
it  is  of  no  importance  what  the  strength  of  the  alkaline 
solution  may  be. 

A  neutral  broth  so  prepared  will  serve  for  the  growth 
of  most  organisms,  but  the  best  growths  are  obtained 
with  a  broth  slightly  acid  to  phenolphthalein.  If  it  be 
desired  to  use  a  less  or  more  alkaline  broth  it  is  necessary 
to  have  an  alkaline  solution  of  known  strength. 

The  solutions  used  are  the  so-called  ''normal  solutions." 
A  normal  solution  is  a  solution  of  the  "  equivalent  " 
weight  in  grammes  of  the  substance  dissolved  in  a  litre 
of  distilled  water.  If  the  metal  of  the  salt  be  monovalent, 
i.e.,  if  it  is  replaceable  in  a  compound  by  one  atom  of 
hydrogen,  the  "  equivalent "  weight  is  the  molecular 
weight  in  grammes.  If  the  metal  be  bivalent,  i.e.,  if  it 
requires  two  atoms  of  hydrogen  to  replace  it  in  a 
compound,  the  equivalent  weight  is  half  the  molecular 
weight  in  grammes.  A  decinormal  solution  is  one-tenth 
of  that  strength  or  one-tenth  of  the  equivalent  weight 
in  grammes  dissolved  in  a  litre  of  water.  A  centinormal 
solution  is  one-hundredth  of  the  same  weight  dissolved 
in  a  litre ;  whilst  a  dekanormal  solution  is  ten  times 
as  strong  as  the  normal,  or  ten  times  the  weight  dis- 
solved in  a  litre.  For  instance,  the  equivalent  weight  of 
sodium  hydrate  NaOH  is  23  +  16  +  1  =  40,  of 
sulphuric  acid  H2S04,  as  it  neutralizes  two  molecules 
of  sodium  hydrate,  is  \  (2  +  32  -+■  64)  or  %*  =  49.  A 
normal  solution  is  represented  by  j,  a  decinormal  by  ~ 
a  centinormal  by  T^.  A  normal  solution  of  sodium 
hydrate  therefore  is  40  grammes  dissolved  in  water  and 
diluted  to  1,000  cc,  whilst  a  normal  solution  of  sulphuric 
acid  will  be  49  grammes  diluted  to  1,000  cc. 

A  neutral  broth  is  one  which  is  neutral  when  tested 
hot  with  phenolphthalein  ;  such  a  broth  is  usually  alkaline 


NEUTRALIZATION  381 

when  tested  by  that  uncertain  standard,  litmus  paper. 
The  degree  of  alkalinity  of  a  broth  is  measured  by  the 
number  of  c.c.  of  normal  alkaline  solution  added  per 
1,000  cc.  of  broth  over  and  above  that  required  for 
neutralization.  The  minus  sign  —  is  used  to  indicate  the 
alkalinity,  so  that  —  4  would  indicate  that  4  c.c.  of  a 
solution  ^  of  alkali  had  been  added  to  1,000  litres  of  the 
broth  in  excess  of  the  amount  required  for  neutralization. 

If  the  broth  used  is  still  acid  as  tested  by  phenol- 
phthalein  that  is  indicated  by  the  plus  sign  4-.  A  broth 
described  as  +  10  would  still  require  the  addition  of 
10  c.c.  of  ^  solution  of  alkali  per  litre  for  neutralization. 
Many  specimens  of  Bovril  broth,  without  neutralization, 
are  not  more  acid  than  this,  and  4-  10  is  a  favourite 
reaction  for  the  growths  of  many  organisms. 

This  question  of  neutralization  and  of  uniformity  of 
reaction  is  a  simple  but  important  matter.  The  degree 
of  alkalinity  or  otherwise  of  the  media  affects  the 
properties  of  growths  so  materially  that  it  is  necessary 
to  be  particular  on  the  point,  but  for  mere  growth  of 
most  organisms  a  broth  neutral  to  phenolphthalein  will 
suffice. 

After  neutralization  or  procuring  the  required  degree  of 
alkalinity  or  acidity  to  phenolphthalein,  the  broth  should 
be  boiled  and  kept  at  the  temperature  of  boiling  water 
for  half  an  hour.  It  should  then  be  allowed  to  cool,  as 
it  is  not  till  it  is  cold  that  the  mass  of  the  phosphates 
will  be  precipitated.  It  is  then,  whilst  cold,  to  be  filtered 
through  ordinary  white  filter  paper.  The  broth  is  now 
prepared,  but  in  the  course  of  the  preparation  many 
organisms  will  have  gained  access  to  it  from  air,  vessels, 
&c,  and  if  left  as  it  is  these  would  multiply.  Steriliza- 
tion is  therefore  necessary.  This  can  either  be  done  in 
bulk  or  the  broth  can  be  decanted  into  a  series  of  test 
tubes  in  quantities  suitable  for  use. 

The  procedure  differs  little  in  the  two  cases.  If  it  be 
desired  to  keep  the  broth  in  bulk  it  should  be  poured 
into   a    clean    narrow-necked    vessel    (Erlenmeyer   flask, 


}82 


STERILIZATION 


tig.  154),  which  will  stand  heat,  and  the  mouth  of  this 
vessel  plugged  tightly  with  non-absorbent  cotton-wool. 
If  it  is  to  be  divided,  some  10  c.c.  should  be  poured  into 
each  of  a  series  of  clean  test  tubes  and  the  mouth  of  each 
should  be  plugged  with  cotton-wool.  It  is  better  to 
sterilize,  by  dry  heat,  the  flask  or  the  tubes  and  wool 
before  pouring  in  the  broth.  This  is  not  absolutely 
essential,  as  the  tubes,  wool,  and  broth  contained  in  the 
tubes  can  all  be  sterilized  together,  but  is  advisable. 

For  sterilization  a  single  boiling  does  not  suffice,  as 


Fig.  154. 


spores  are  only  slowly  killed  at  the  temperature  of  boiling 
water. 

Sterilization. — To  sterilize,  the  broth  and  the  vessels 
■containing  it  should  be  maintained  at  the  temperature 
of  boiling  water  for  at  least  half  an  hour  on  three 
consecutive  days  and  allowed  to  cool  in  between.  This 
intermittent  method  allows  the  spores  which  have 
escaped  the  first  sterilization  to  develop  into  the  less 
resistant  organisms  before  the  second  heating,  which 
then  destroys  them.  The  third  sterilization,  which  is 
not  always  absolutely  necessary,  is  a  precaution  in  case 
any  spores  or  organisms  have  escaped  from  the  two 
previous  sterilizations. 

Storing  Media. — The  broth  when  cool  is  ready  for 
use  and  can  be  kept  till  required.  The  tubes,  wool,  and 
broth  are  all  sterile   and    remain   so    for  a  considerable 


VARIOUS    MEDIA  383 

period.  Organisms  can  only  gain  access  to  the  broth 
by  growing  through  the  wool.  This  does  not  take  place 
through  dry  wool,  but  in  moist,  warm  climates,  such  as 
are  met  with  in  the  Tropics,  the  wool  gets  damp  and 
growth  through  it  takes  place. 

In  such  climates  it  is  advisable  as  a  routine  every 
week  to  heat  the  end  of  the  test  tube  containing  the 
cotton-wool  so  as  to  ensure  the  wool  being  dry  and 
to  kill  any  organisms  that  have  grown  in  it.  Unless  this 
precaution  is  taken  the  tubes  soon  become  contaminated. 
In  a  moist  climate  tubes  pept  in  a  cool  incubator  become 
contaminated  so  rapidly  that  the  cotton  wool  should  be 
heated  daily. 

The  number  of  organisms  falling  on  to  the  cotton- 
wool can  be  greatly  reduced  by  covering  the  top  of  the 
tube  with  an  inverted  paper  cone,  such  as  a  folded  filter 
paper.  This  measure  would  delay  the  contamination  of 
wool  even  in  the  Tropics. 

Glycerine  Broth,  &c. — For  many  purposes  additions 
are  made  to  the  nutrient  broth.  These  additions  must 
be  made  before  neutralization  and  sterilization ;  if  made 
after,  the  sterilizations  will  require  to  be  repeated  and  the 
neutralization  readjusted. 

Glycerine  broth  is  made  by  the  addition  of  6  per  cent, 
of  glycerine.  Glucose,  lactose,  maltose  or  saccharose, 
added  in  the  proportion  of  2  per  cent,  to  the  broth, 
make  glucose  broth,  lactose  broth,  &c,  respectively. 

Solid  Media — The  broth  is  mixed  with  gelatine  or 
agar-agar  in  sufficient  proportion  for  the  solution  to  set 
when  cooled  to  the  temperatures  at  which  it  is  desired 
to  study  the  growths. 

Nutrient  Gelatine. — The  gelatine  medium  is  made  by 
the  addition  of  9  to  12  per  cent,  of  the  best  French 
gelatine  to  the  crude  broth.  Broth  that  has  been 
neutralized  and  filtered  can  be  used,  but  it  is  waste  of 
time,  as  neutralization  will  have  to  be  repeated.  Gelatine 
is  always  acid. 

After  the  gelatine  has  been  added  in  the  required 
proportion  keep  in  the  steamer  for  half  an  hour  ;    neu- 


384  SOLID   MEDIA 

tralize,  render  alkaline,  or  leave  acid  to  the  required 
extent.     Allow  to  cool  to  450  C. 

Whip  up  the  white  of  an  egg  for  each  500  cc.  of  the 
gelatine  broth  and  mix  well  with  the  medium.  Steam 
for  half  an  hour.  The  white  of  the  egg  diffused  through 
the  medium  will  coagulate,  and  in  its  coagulation  will 
carry  down  many  of  the  impurities  of  the  gelatine. 
Filter  whilst  hot  through  a  coarse  filter  paper — Chardin's 
— which  should  be  moistened  with  hot  water.  Store  in 
flasks  or  decant  into  test  tubes  as  required.  Sterilize  as 
with  nutrient  broth  for  half  an  hour  on  three  consecutive 
days.  This  medium  is  known  as  nutrient  gelatine,  or 
simply  "gelatine." 

Glucose,  &c,  &c,  can  be  added  to  it  if  required,  the 
addition  being  made  preferably  before  neutralization  and 
always  before  sterilization.  Too  prolonged  heating  causes 
hydrolytic  changes  in  the  gelatine,  so  that  it  will  not  set. 
Extra  sterilizations  must  be  avoided  where  possible  on 
this  account. 

Nutrient  Agar. — Agar  or  nutrient  agar  is  made  in  a 
similar  manner;  1*5  to  2  per  cent,  of  the  powered  agar 
is  added  instead  of  the  gelatine.  It  is  much  more 
difficult  to  filter,  and 'where  the  necessary  time  cannot 
be  given,  a  passable  substitute  is  to  allow  it  to  cool  slowly 
so  as  to  permit  the  coagulated  egg  albumin  and  other 
precipitates  to  settle  to  the  bottom.  When  cold  the 
mass  can  be  removed  from  the  vessel  and  the  lower  part 
containing  the  great  mass  of  the  impurities  cut  off.  The 
residue,  though  much  inferior  in  appearance  to  the 
filtered  product,  is  sufficiently  clear  to  be  translucent 
and  can  be  satisfactorily  used  without  filtration  for 
cultures. 

The  clearer  filtered  product  is  better.  To  filter  it  is 
necessary  that  the  filter  paper  should  be  kept  hot.  This 
is  best  effected  by  placing  the  funnel,  filter  paper,  and 
receptacle  in  the  steam  sterilizer  and  allowing  the  filtra- 
tion to  take  place  in  the  steam  sterilizer. 

Filtration  of  these  media  is  facilitated  by  folding  the 


SEPARATION    OF   ORGANISMS  385 

filter  paper  so  as  to  have  a  large  number  of  angles  and 
very  little  of  the  paper  in  contact  with  the  glass.  These 
papers  can  be  bought  ready  folded  or  can  be  folded 
before  use. 

Addition  of  glucose,  &c,  can  be  made  as  in  the  case 
of  other  media  before  neutralisation  and  filtration. 

The  solid  media  are  essential  for  the  separation  of 
the  various  organisms  usually  present  in  the  animal 
tissues,  discharges,  or  other  substances  to  be  examined. 

Separation  of  Organisms. — The  method  of  procedure 
is  based  on  the  principle  that  by  successive  dilutions 
of  a  minute  quantity  of  the  substance  to  be  examined 
the  individual  organisms  will  be  so  scantily  distributed 
through  the  medium  that  they  will  be  separated  from 
each  other  by  an  interval  appreciable  to  the  eye.  If 
this  medium  be  then  allowed  to  become  solid  the  or- 
ganisms will  remain  well  separated  from  each  other, 
and  if  kept  under  conditions  favourable  for  their  growth 
will  multiplv  and  form  in  the  course  of  a  few  days 
"colonies"  which  will  be  visible  to  the  naked  eye. 
From  these  colonies  sub-cultures  can  be  made,  and  the 
colonies  may  also  be  examined  directly. 

A  piece  of  platinum  wire,  3  to  4  in.  in  length,  is 
inserted  into  the  fused  end  of  a  glass  rod,  and  a  small 
loop  is  made  at  the  other,  the  free  end,  of  the  platinum 
wire.  This  wire  is  sterilized  by  heating  in  the  flame, 
and  a  loopful  of  the  substance  to  be  examined  is  taken 
up  by  this  loop. 

A  tube  containing  the  gelatine  medium  melted  by 
placing  in  hot  water,  of  a  temperature  not  exceeding 
280  C,  is  then  inoculated  with  this  loopful,  and  the 
tube  is  rolled  between  the  hands  to  secure  uniform  ad- 
mixture. 

The  amount  of  the  substance  is  thus  diluted  by  the 
amount  of  the  fluid  gelatine. 

After  sterilizing  the  needle  a   loopful  from    this  tube 
is  inoculated  into  a  second  tube  and  will  again  be  diluted 
to  the  same  extent.     A  third  tube  is  treated  in  the  same 
manner  and  the  dilution  will  now  be  extreme. 
25 


386  PLATING 

In  other  words,  provided  the  mixing  is  thorough  the 
organisms  will  be  so  much  diluted  by  these  successive 
dilutions  that  they  will  be  separated  from  each  other  by 
appreciable  intervals.  A  fourth  or  fifth  dilution  may 
be  made,  but  it  is  not  usually  required,  as  the  third  dilu- 
tion is  in  most  instances  sufficient. 

The  end  of  each  of  these  tubes,  with  the  plug  with- 
drawn, is  heated  in  turn  to  destroy  any  organisms  which 
may  be  present  at  the  end  of  the  tube  and  the  gela- 
tine is  poured  into  a  flat  sterilized  glass  dish  (fig.  155) 
— a  Petri  dish — which  is  quickly  covered  with  another 
similar  but  larger  sterilized   dish.      The  melted  gelatine 


Fig.  155. 

solidifies  as  a  thin  sheet  of  nutrient  gelatine,  and  is 
allowed  to  remain  at  a  temperature  of  about  20°  to 
22°  C. 

Some  organisms  will  grow  quickly  and  others  slowly, 
and  by  colour,  size,  shape  of  colonies  and  effect  on  the 
gelatine  it  is  usually  possible  to  distinguish  that  several 
organisms  are  present.  In  the  plate  from  the  first  tube 
the  colonies  are  so  numerous  that  they  are  separated 
from  each  other  by  too  small  a  distance  to  isolate.  In 
the  plate  from  the  second  the  organisms  may  be  suffi- 
ciently far  apart,  and  in  the  third  and  subsequent 
dilutions  the  colonies  resulting  from  the  growth  of  the 
widely  separated  organisms  are  usually  far  enough  apart 
to  be  easily  distinguished  from  each  other.  From  these 
cultures  can  be  made. 

As  the  first  dilution  is  too  little  diluted  for  practical 
work  and  the  second  is  usually  so,  it  is  unnecessary  to 
make  these  dilutions  in  the  solid    medium  or  to   make 


PLATING  387 

plates  of  them.  The  two  first  dilutions  may  be  done  in 
sterile  broth  or  even  in  a  weak  sterile  salt  solution, 
5  grm.  to  a  litre,  and  only  the  third  in  the  solid 
medium.  This  economizes  the  solid  medium,  which  is 
the  most  troublesome  to  prepare. 

Plating  may  be  done  with  agar,  but  a  thermometer 
must  be  used  to  make  sure  that  the  agar  is  cool  enough, 
as  if  too  hot  the  organisms  may  be  killed.  The  agar  will 
have  to  be  heated  to  nearly  the  boiling  point  of  water  to 
become  thoroughly  fluid,  and  allowed  to  cool  to  about 
440  C.  before  inoculation.  It  is  not  so  easy  a  proceeding 
as  plating  with  gelatine,  but  agar  is  the  only  solid  medium 
that  can  be  used  in  many  parts  of  the  Tropics,  as  above 
220  C.  the  gelatine  will  not  set.  Stronger  solutions,  as 
20  per  cent,  gelatine,  will  remain  solid  at  37. 50  C,  but 
these  stronger  gelatines  are  not  easy  to  work  with  and 
frequently  undergo  changes  during  sterilization  that  cause 
liquefaction  or  acid  production.  Unless  ice  and  a  cold 
incubator  are  available  we  are  restricted  to  the  use  of 
agar  for  plating. 

A  convenient  method  of  plating  on  agar  is  to  make 
the  agar  plates  and  inoculate  when  the  agar  has  set 
either  from  the  second  or  third  broth  solution,  by  making 
a  series  of  parallel  strokes  with  a  platinum  loop  on  the 
solidified  surface,  or,  and  better,  by  using  a  sterilized 
brush — camel's  hair — and  brushing  lightly  over  the  sur- 
face of  the  medium  after  dipping  this  brush  in  the  second 
or  third  broth  dilution.  Excess  of  fluid  is  to  be  avoided 
by  draining  off  from  the  brush  against  the  inner  side  of 
the  tube.  The  brush  should  be  sterilized  in  a  dry  tube, 
plugged  with  wool,  by  three  successive  sterilizations. 
The  platinum  wire  is  sterilized  as  usual  by  heating  in 
the  flame. 

Some  important  organisms  will  not  grow  on  any 
known  artificial  medium,  and  others  only  on  special 
media  or  under  special  conditions.  Separation  of  such 
organisms  is  either  impossible  or  difficult.  Standard 
books  on  bacteriology  should  be  consulted  for  methods, 


388  DESCRIPTION   OF   ORGANISMS 

but  these  will  not  usually  be  practicable  for  the  solitary 

practitioner    under    the    conditions    of    tropical    life    and 
work. 

Description  of  Organisms. — Having  obtained  a  pure 
culture  of  an  organism  the  more  important  points  to 
determine  are  as  follows  : — 

(1)  Size,  shape  and  arrangement.  Morphological 
appearance. 

(2)  Motility. 

(3)  Spore  formation. 

(4)  Structure.     Flagella,  capsule,  &c. 

(5)  Staining  reactions  :  (a)  Simple  stains  ;  (h)  Gram's 
method  ;   (c)  Ziehl-Neelson. 

(6)  Growths  on  artificial  media:  (a)  In  broth;  (b) 
on  gelatine  ;  (c)   on  agar. 

(7)  Conditions  :  (a)  Essential  to  growth  ;  (/>)  favour- 
able to  growth  ;   (c)  inimical  to  growth. 

(8)  Chemical  products  :  Gas  formation  and  curdling 
of  milk  ;  acid  or  alkali  formation  ;  indol  formation. 

(9)  Reaction  with  blood  sera,  particularly  with  the 
blood  sera  of  patients  suffering  from  definite  diseases. 

(10)  Pathogenic  properties. 

In  organisms  such  as  B.  lepra',  which  are  cultivated 
with  great  difficulty,  only  a  few  of  these  points  can  be 
determined  ;  and  the  pathogenicity  has  not  been  proved 
experimentally,  as  with  doubtful  exceptions,  lower  animals 
are  insusceptible.  The  causal  relation  is  inferred  from  the 
constant  association  of  the  organisms,  identified  by  their 
staining  reactions  and  appearances  in  the  lesions  resulting 
from  the  disease,  and  from  the  observation  that  they  are 
not  found  in  man  except  in  this  disease. 

We  propose  to  take  briefly  the  methods  of  observing 
these  various  points.  The  descriptive  terms  used  with 
reference  to  them,  the  relative  value  and  the  limitations 
to  the  value  of  each  point  for  diagnostic  purposes  will 
also  be  considered. 

(1)  The  size  and  shape  of  an  organism  is  best  observed 
in    stained    specimens,   and    any   simple    stain    combined 


PREPARATION    OK    FILMS  389 

with  a  mordant  will  suffice.  Films  should  be  made  on 
a  slide  or  cover-glass.  For  this  purpose  with  a  culture 
in  broth  all  that  is  required  is  to  spread  a  drop  with  the 
platinum  wire.  If  the  culture  is  on  agar  or  gelatine  it 
should  be  rubbed  up  with  a  little  water  (sterile),  and  of 
this  emulsion  a  portion  should  be  spread  out  with  a 
needle. 

Preparation  of  Films. — If  it  be  desired  to  make  a  film 
or  smear  from  any  natural  fluid  or  excretion  it  may 
require  the  addition  of  a  little  water  to  make  a  satis- 
factory smear.  From  blood  thick  films  may  be  used 
and  decolorized  by  removing  the  haemoglobin  with 
sterile  water.  Tissues  should  be  cut  with  a  sterile  knife 
and  the  cut  surface  rubbed  on  the  slide. 

The  most  difficult  films  to  make  are  those  such  as 
sputum  containing  much  mucus.  The  most  satisfactory 
method  is  to  use  two  slides  and  warm  both.  A  portion 
of  the  mucus  is  transferred  to  one  slide  and  this  is 
warmed  over  a  Bunsen  flame  or  spirit  lamp,  and  a 
second  slide  is  warmed  at  the  same  time.  The  second 
warmed  slide  is  used  to  rub  the  mucus  on  the  first  and 
is  placed  with  the  long  axis  at  right  angles  to  the 
other,  and  the  surfaces  parallel  and  in  contact.  The 
slides  are  then  separated  and  both  are  again  warmed  in 
the  flame  with  the  smeared  side  of  each  uppermost. 
They  are  again,  whilst  still  hot,  rubbed  together  with  the 
smeared  surface  of  the  two  in  contact.  This  process  is 
repeated  till  the  films  are  nearly  dry,  when  they  are 
finally  rubbed  together  harder.  Good  thin,  dry  films 
are  easily  and  quickly  obtained  by  this  method. 

However  the  films  are  made  they  require  fixation. 
This  is  best  done  by  heat  and  is  usually  accomplished 
by  passing  through  a  smokeless  flame  three  times,  the 
smeared  side  always  uppermost.     Do  not  char  the  film. 

As  many  films  are  so  thin  that  they  are  difficult  to 
see  when  dry,  it  is  well  to  mark  the  smeared  side  of  the 
slide  with  a  grease  pencil.  The  staining  fluid  is  simply 
placed    over   the   film    for    the   requisite   time   and   then 


39°  SCHIZOMYCETES 

washed  off.  Stains  used  are  Ldffler's  blue,  five  to  ten 
minutes  ;  carbol  thionin,  five  minutes  ;  carbol  fuchsin 
(i — 4)  or  gentian  violet  for  organisms  that  do  not  take 
other  stains  deeply,  one  to  ten  minutes. 

SCHIZOMYCETES. 

Bacteria  or  Schizomycetes  are  unicellular  vegetable 
organisms.  Reproduction  is  by  fission.  Resistant  forms 
called  "  spores  "  may  be  produced. 

They  are  differentiated  by  these  characters  from 
Hyphomycetes  or  moulds,  in  which  spores  form  in 
specially  differentiated  cells,  and  from  Blastomycetes  or 
yeasts,  which  are  oval  or  rounded  bodies,  and  in  which 
reproduction  is  accomplished  by  budding. 

( i )  Morphology. — The  various  shapes  of  bacteria  usually 
described  are  Cocci  or  rounded  or  oval  bodies,  with  the 
greatest  diameter  not  more  than  twice  the  least.  The  term 
Micrococci  is  used  for  the  smaller  forms.  If  division  takes 
place  only  in  one  direction  the  organisms  may  remain 
attached  in  pairs,  Diplococci,  or  in  chains,  Streptococci,  if  a 
series  remain  attached. 

In  other  cases  division  takes  place  in  two  directions, 
and  we  then  find  the  organisms  arranged  in  squares  of 
four  or  multiples  of  four.  Such  growths  are  called 
Tetrads.  Others  divide  in  three  directions  at  right  angles 
to  each  other  and  form  cubical  masses,  these  are  known 
as  Sarcincv. 

A  common  arrangement  is  irregular  growth  in  all 
directions,  leading  to  an  irregular  mass  or  cluster  of 
cocci,  Staphylococci. 

Bacilli  are  cells  that  are  rod-shaped.  They  are  longer, 
at  least  twice  as  long  as  they  are  broad,  and  the  shorter 
forms  are  distinguished  from  oval  cocci  by  having  the 
two  sides  parallel.  By  fission  they  may  grow  into  long 
jointed  rods — streptobacilli. 

Curved  organisms  are,  when  short,  known  as  Vibrios, 
when  long  and  more  twisted  as  Spirilla  or  Spirobacteria. 


MOTILITY  391 

Leptothrix. — Rod-shaped,  filamentous  forms  showing 
differentiation  between  base  and  apex,  but  not  branching. 

Streptothrix. — Filamentous  forms  showing  true  branch- 
ing. These  form  the  connecting  link  between  the  Schizo- 
mycetcs  and  the  Hyphomycetes. 

Measurements  are  made  as  for  other  minute  bodies. 
Some  organisms  readily  change  their  form  with  varia- 
tions in  the  condition  under  which  growth  has  taken 
place.  If  the  variations  are  great  the  organism  is  de- 
scribed as  "  pleomorphic."  Slight  variations  occur  in  all 
organisms,  so  that  morphological  characters  alone  are 
not  to  be  relied  on. 

(2)  Motility.—  This  can  only  be  observed  in  living 
cultures,  though  it  can  be  inferred  for  organisms  which 
are  shown  to  have  flagella.  The  motion  of  motile  organ- 
isms must  be  clearly  distinguished  from  the  oscillatory 
movement — Brownian  movement — common  to  all  minute 
particles  suspended  in  fluid. 

True  motility  is  best  observed  in  a  "  hanging  drop " 
preparation.  This  is  made  by  making  a  thick  ring  with 
vaseline  on  a  slide  and  taking  a  clean  cover-glass,  rather 
larger  than  this  ring,  and  placing  near  the  centre  a  small 
drop  of  the  culture  of  living  organisms  to  be  examined. 
The  slide  is  then  taken  up  and  turned  so  that  the  vaseline 
ring  is  directed  downwards,  and  is  gently  brought  into 
contact  with  the  cover-glass  so  that  the  drop  of  culture 
on  the  cover  is  in  the  centre  of  the  ring  of  vaseline. 
The  cover  will  adhere  to  the  vaseline  ring  and  form  a 
sealed  chamber,  and  when  the  slide  is  turned  over  again 
the  drop  will  hang  from  the  lid  of  this  chamber,  the 
cover-glass,  and  can  then  be  examined.  If  the  tempera- 
ture is  so  high  that  the  vaseline  runs,  lard  can  be 
substituted  for  it. 

The  organisms  are  colourless  and  transparent  and 
difficult  to  focus,  so  that  the  light  must  be  reduced  by 
nearly  closing  the  iris  diaphragm.  Either  ^  or  ^2  °^ 
immersion  objective  may  be  used.  It  is  well  to  focus 
first  on  to  the  edge  of  the  vaseline  ring  and  then  move 


392  SPOKE    FORMATION 

the  slide  towards  the  drop,  keeping  the  droplets  of  water 
of  condensation  which  usually  form  on  the  under  surface 
of  the  cover-glass  in  focus  till  the  edge  of  the  drop  is 
reached.  With  a  little  practice  and  a  dim  light  the 
organisms  can  then  be  brought  into  focus  and  the  pres- 
ence or  absence  of  automatic  motility  determined. 

This  property,  though  an  important  point  of  difference 
between  some  organisms  that  closely  resemble  each  other 
morphologically,  is  subject  to  considerable  variation,  and 
the  degree  of  motility  in  motile  organisms  varies  from 
slight  causes,  such  as  slight  difference  in  temperature, 
reaction  of  the  medium,  &c. 

(3)  Spore  Formation. — All  the  micro-organisms  repro- 
duce by  fission,  but  some  of  them  also  enter  into  a 
resting  stage — spores.  The  resting  form  is  much  more 
resistent  to  agencies,  chemical,  heat,  &c,  which  destroy 
organisms,  so  that  spores  will  withstand  for  some  time 
the  temperature  of  boiling  water,  though  the  active  phase 
of  the  organism  is  at  once  destroyed. 

Some  organisms  form  spores  very  readily,  others  only 
under  certain  circumstances  not  thoroughly  understood, 
and  many  pathogenic  and  other  organisms  do  not  form 
spores  under  any  known  circumstances. 

The  spores  can  often  be  recognized  in  the  living  culture, 
as  they  are  usually  more  highly  retractile  as  well  as 
rounder.  For  the  demonstration  in  dried  films  advantage 
is  taken  of  the  fact  that  spores  stain  with  greater  difficulty, 
but  when  stained,  retain  their  stain  better  than  the  organ- 
isms from  which  they  were  derived.  A  simple  method 
is  to  stain  with  warm  carbol  fuchsin  for  five  minutes. 
This  much  overstains  both  spores  and  bacilli.  Treat 
rapidly  with  2  per  cent,  sulphuric  acid  ;  this,  if  done 
rapidly,  will  leave  the  spores  stained,  but  remove  the 
stain  entirely  from  the  bacilli.  Wash  well  to  remove 
the  last  traces  of  acid  and  counter-stain  with  Loffler's 
blue  for  ten  minutes,  or  carbol  thionin.  The  spores  will 
be  stained  by  the  fuchsin  and  the  bacilli  blue  by  the 
methylene  blue  or  thionin. 


STRUCTURE   OF   ORGANISMS  393 

This  method  is  successful  for  most  spore-forming 
organisms. 

(4)  Structures  of  Organisms. — Certain  points  in  the 
structure  of  organisms  are  sufficiently  definite  to  be  of 
use  in  diagnosis. 

Capsules. — Some  organisms  have  a  thick  capsule  which 
does  not  stain  deeply  with  basic  stains,  or  may  not  stain 
at  all.  The  simplest  method  of  demonstration  is  to  stain 
the  film  with  carbol  fuchsin  and  to  examine  in  -water,  not 
in  Canada  balsam.  The  organism  is  surrounded  by  a 
clear  space,  and  the  capsule  with  a  defined  edge  can 
usually  be  seen. 

Welch  treats  the  film  with  2  per  cent,  acetic  acid,  which 
causes  the  capsule  to  swell  and  enables  it  to  take  the 
stain,  and  then  after  removal  of  the  acid  he  stains  with 
aniline  gentian  violet  for  five  to  thirty  seconds.  Cap- 
sulated  organisms  often  lose  their  capsules  in  culture,  but 
the  presence  or  absence  of  a  capsule,  as  seen,  for  instance, 
in  sputum,  is  of  value. 

A  cell  wall  is  probably  present  in  all  the  organisms, 
but  it  is  difficult  to  demonstrate.  In  some,  however,  it 
is  fairly  well  marked.  It  is  best  shown  after  the  cell 
contents  have  been  caused  to  shrink  by  salt  solutions 
or  iodine  solution  (plasmolysis). 

Flagella.— Motile  organisms  have  been  shown  to  have 
flagella.  They  are  variable  in  number,  and  whilst  the 
vibrios  have  usually  only  one  or  two  the  motile  bacilli 
may  have  large  numbers.  The  number  of  flagella  is  of 
some  value  in  the  differentiation  of  species,  and  the  pres- 
ence, absence,  or  plan  of  arrangement  is  of  differential 
value  in  grouping  organisms. 

The  methods  of  demonstration  cannot  be  considered 
as  satisfactory  or  easy,  and  there  is  considerable  uncer- 
tainty in  the  results  ;  they  are  all  troublesome.  The 
two  common  methods  successfully  employed  are  Muir's 
modified  Pitfield  and  MacCrorie's. 

By  Muir's  method  the  mordant  employed  is  composed 
of:— 


394  FLAGELLA   STAINING 

Tannic  acid  to  per  cent,  aqueous  solu- 
tion            io  c.c. 

Corrosive   sublimate   saturated    aqueous 

solution  ...  ...  ...  ..        5  c.c. 

Alum  saturated  aqueous  solution  ...       5  c.c. 

Carbol  fuchsin      ...  ...  ....         ...        5  c.c. 

This  is  well  mixed,  allowed  to  settle,  and  the  clear 
fluid  decanted  off  and  centrifugalized.  This  mordant 
keeps  for  about  a  fortnight,  but  must  be  centrifugalized 
each  time  before  use. 

The  stain  employed  is  composed  of  a  saturated  solution 
of  alum,  25  c.c,  with  5  c.c.  of  alcoholic  gentian  violet 
saturated  solution.  This  must  be  prepared  immediately 
before  use. 


Fig.  156. 

The  smears  should  be  made  from  agar  cultures,  twelve 
to  eighteen  hours  old,  emulsified  with  a  little  distilled 
water.  Spread  very  gently  on  a  cover-glass,  freshly 
flamed  to  free  from  grease.  Hold  in  Cornet's  forceps 
(fig.  156),  being  careful  that  the  film  side  corresponds  to 
the  fenestrated  side  of  the  forceps,  otherwise  mistakes  as 
to  which  is  the  film  side  may  occur. 

Allow  the  film  to  dry  in  air  and  fix  by  passing  through 
the  flame.  Cover  with  the  mordant  and  heat  till  it 
steams  for  two  minutes.  Wash  well  in  water  and  dry 
carefully.  Pour  on  the  gentian  violet  stain  and  heat 
till  the  staining  fluid  steams  for  two  minutes.  Wash 
in  water,  dry  and  mount  in  Canada  balsam. 

This  method  requires  the  use  of  a  centrifuge,  but  gives 
a  large  proportion  of  successful  results. 

MacCrorie's  method  is  simpler.  A  single  stain  com- 
bined with  a  mordant  is  used  for  staining  the  flagella 
and  the  bacilli  are  counter-stained  with  carbol  fuchsin. 


SIMPLE   STAINS  395 

The  stain  is  composed  of : — 

Night  blue  saturated  alcoholic  solution     10  c.c. 
Potash  alum  saturated  aqueous  solution    10  c.c. 
Tannin  10  per  cent,  aqueous  solution...      10  c.c. 
Gallic  acid  i  or  2  grin,  improves  the  solution. 
Films  from  young  agar  cultures  are  prepared  as  above 
and  the  stain  is  placed  on  the  film  for  five  minutes  and 
slightly  warmed.     It  must  be  flushed  off   with  running 
water  or  a  thick,  dirty  deposit  will  be  left.     Counter-stain 
with  carbol  fuchsin,  dry  and  mount. 

(5)  Differentiation  by  Methods  of  Staining. — There  are 
three  main  methods  of  diagnostic  value  : — ■ 

(a)  Simple  Stains. — There  is  great  variation  in  the 
ease  with  which  different  organisms  take  up  stains,  and 
this  difference  is  sometimes  of  value.  Some  organisms 
do  not  stain  uniformly,  and  such  differences  as  pre- 
ferential affinity  of  stains  for  the  ends  of  a  bacillus, 
bipolar  staining,  is  one  of  the  characteristics  of  the 
plague  bacillus.  In  cultures  organisms  frequently  lose 
their  characteristic  staining  reactions. 

Of  greater  value  are  two  special  methods. 

(b)  Gram's  Method  is  based  on  the  fact  that  some 
organisms  will  retain  their  stain  when  treated  with  alcohol 
if,  after  staining,  they  are  treated  with  a  solution  of  iodine. 
A  freshlv  prepared  solution  of  gentian  violet  in  aniline 
water  is  made  by  shaking  up  a  few  drops  of  aniline  oil 
with  water  and  filtering.  To  this  is  added  drop  by  drop 
a  saturated  alcoholic  solution  of  gentian  violet  till  a 
metallic  film  begins  to  form  on  the  surface.  With  this 
stain  the  fixed  film  is  stained  for  five  minutes.  Carbolic 
acid  1  in  20  can  be  used  instead  of  the  aniline  water.  If 
now  treated  with  alcohol  the  stain  would  be  completely 
removed  from  all  the  organisms. 

In  some  organisms  the  addition  to  the  film  of  Gram's 
iodine  solution,  composed  of  iodine  1  part,  potassium 
iodide  2  parts,  and  water  300  parts,  for  two  minutes, 
will  fix  the  stain  in  these  organisms  so  that  when  the 
film  has  been  treated  with  alcohol   they  still  retain  the 


396  GRAM'S    .METHOD 

purple  colour,  whilst  it  is  removed  from  everything  else. 
The  organisms  which  retain  their  stains  are  those  which 
are  described  as  "staining  by  Gram." 

The  alcohol  is  kept  on  till  it  ceases  to  remove  any  more 
colour  and  not  longer,  as  in  time  it  will  remove  the  stain 
even  from  the  organisms  which  stain  by  Gram. 

It  is  convenient  instead  of  using  a  plain  alcohol  to 
use  an  alcoholic  solution  of  eosin  i  per  cent.,  as  then 
organisms  which  do  not  stain  by  Gram  will  he  stained 
faintly  by  the  eosin.  This  is  of  most  use  when  working 
with  a  mixture  of  organisms,  such  as  is  met  with  in  many 
secretions,  &c. 

For  sections  it  has  the  additional  advantage  that  it 
does  not  require  such  prolonged  treatment  with  alcohol 
as  is  required  if  alcohol  is  first  used  to  decolourize,  and 
then  again  for  dehydrating  after  counter-staining. 

As  the  action  of  alcohol  is  so  rapid  when  working 
with  organisms  that  retain  the  stain  less  firmly,  another 
agent  that  decolourizes  more  slowly  is  better.  The 
agent  used  is  aniline  oil.  This  decolourizes  the  non- 
Gram  staining  organisms  as  effectually  as  alcohol.  It  is 
used  in  the  same  way  but  can  be  left  on  longer.  If 
counter-staining  is  desired  the  counter-stain  must  be 
used  before  staining  by  Gram. 

The  use  of  aniline  oil  instead  of  alcohol  is  better  for 
sections.  Counter-staining  with  eosin  or  Bismarck  brown 
should  be  done  first.  After  staining  with  Gram  and 
adding  the  iodine,  blot  and  treat  with  aniline  oil.  This 
will  dehydrate  as  well  as  remove  the  stain  from  non- 
Gram  staining  organisms.  Then  wash  off  the  aniline  oil 
with  xylol  and  mount  in  xylol  balsam. 

Most  of  the  pyogenic  cocci,  the  organisms  usually  found 
in  suppuration,  stain  by  Gram.  Many  of  the  organisms 
associated  with  intestinal  and  other  diseases  do  not  stain 
by  Gram. 

(c)  ZlEHL-NEKLSON's  METHOD. — A  comparatively  small 
number  of  groups  of  organisms  are  described  as  acid  fast, 
because  when  once  stained  they  retain  the  stain  even  after 


ZIEHL-NEELSON'S    METHOD  397 

treatment  with  fairly  strong,  25  per  cent.,  solutions  of  the 
mineral  acids.  Hydrochloric,  nitric  or  sulphuric  acids 
are  those  used. 

The  method  employed  is  to  use  a  strong  basic  stain 
such  as  fuchsin  in  a  1  in  20  aqueous  solution  of  carbolic 
acid,  and  either  to  stain  in  the  cold  for  some  hours,  or 
more  conveniently  to  warm  until  the  carbol  fuchsin 
steams,  and  then  keep  warm  for  five  minutes. 

This  is  conveniently  done  on  the  slide.  The  film  is 
fixed  as  usual  and  covered  with  the  carbol  fuchsin.  The 
slide  is  placed  on  a  copper  which  has  been  warmed  in 
the  flame  and  left  there,  fresh  stain  being  added  if  evapor- 
ation is  too  rapid  or  the  stain  shows  signs  of  boiling. 

The  stain  is  flushed  off  and  replaced  by  a  25  per  cent, 
solution  of  sulphuric  acid.  The  pink  colour  disappears 
and  is  replaced  by  a  yellow.  The  film  is  again  washed 
and  if  still  pink  again  treated  with  sulphuric  acid.  This 
is  repeated  till  on  washing  at  the  most  a  faint  pink  colour 
returns. 

The  specimen  is  well  washed  in  water  to  completely 
remove  the  acid  and  counter-stained  with  Loffler's  blue 
for  five  minutes.  Wash,  dry,  and  either  examine  directlv 
by  placing  a  drop  of  oil  on  the  film  or  mount  in  Canada 
balsam.  The  acid-fast  organisms  retain  the  red  colour 
of  the  fuchsin,  whilst  other  organisms  are  stained  blue 
by  the  methylene  blue  which  is  used  as  the  counter-stain. 

In  tropical  work  it  is  important  only  to  use  fresh 
carbol  fuchsin.  The  solution  keeps  well  in  England, 
but  in  the  Tropics  it  deteriorates,  so  that  sometimes  in 
a  week  or  so,  and  at  others  in  some  months,  it  ceases  to 
stain  well.  Colour-blind  people  will  find  it  well  to  use 
gentian  violet  instead  of  fuchsin.  A  saturated  alcoholic 
solution  of  gentian  violet  is  added  to  the  1  in  20  carbolic 
acid  to  make  the  stain,  and  Bismarck  brown  is  used  as  the 
counter-stain. 

The  more  important  members  of  the  acid-fast  group 
cannot  be  cultivated  on  the  simple  media.  It  will 
therefore  be  convenient  to  consider  these  organisms  here. 


398  TUBERCLE 

There  are  four  main  groups  of  the  acid-fast  organisms, 
which  will  be  considered  under  the  heading  of  the  best 
known  member  of  the  group  : — 

Tubercle  ;  lepra  ;  smegma  ;  Timothy  grass. 
Some  forms  of  the  Streptothrix  group  are  also  "acid- 
fast." 

The  tubercle  group  includes  the  organisms  found 
in  tuberculosis,  in  mammals,  birds  and  reptiles. 

The  organisms  can  be  cultivated  on  blood  serum 
and  nutrient  glycerine  agar,  or  in  glycerine  veal 
broth.  Growth  is  slow  and  much  affected  by  the 
temperature.  The  preferential  temperature  is  that  of 
the  animal  from  which  the  organisms  were  obtained. 
The  mammalian,  avian  and  reptilian  tubercle 
bacilli  therefore  grow  at  different  temperatures  and 
are  pathogenic  to  mammals,  birds  and  reptiles  re- 
spectively. Some  authorities  hold  that  they  are 
modifications  of  one  and  the  same  organism  and 
that  they  can,  by  suitable  methods,  have  their 
characters  altered  so  that  the  differences  disappear. 
By  most  authorities  the  three  are  considered  to  be 
specifically  distinct,  and  some  go  further  and  do 
not  admit  the  specific  unity  of  the  tubercle  organ- 
isms in  different  mammals.  Koch  holds  that  bovine 
and  human  tuberculosis  are  distinct,  on  the  ground 
that  their  pathogenicity  varies. 

Tubercle  bacilli  in  man  are  found  in  the  secre- 
tions or  excretions  from  an  infected  organ  and  there- 
fore may  be  found  in  sputum,  urine,  &c.  They 
usually  set  up  a  granulomatous  new  growth  which 
has  a  marked  tendency  to  caseate  and  break  clown, 
The  organisms  may  be  present  in  large  numbers, 
but  in  some  situations,  such  as  the  skin,  bones, 
pleural  effusions,  &c,  they  are  usually  found  only  in 
small  numbers.  They  are  found  but  rarely  in  the 
blood. 

The  Lepra  Bacillus  is  the  only  representative  known 
of  this  group.     It  can  be  cultivated  on  artificial  media 


LEPROSY  399 

only  under  very  special  conditions,  and  experiments 
at  inoculation  of  lower  animals  have  usually  failed, 
except  with  these  doubtful  cultures.  The  organ- 
isms are  found  in  extraordinary  numbers  in  leprous 
tubercles  in  the  skin,  and  when  these  ulcerate,  in  the 
discharges  from  these  ulcers.  Before  ulceration  the 
bacilli  can  be  readily  demonstrated,  by  compression 
with  a  clamp  of  a  tubercle  or  portion  of  infiltrated  skin. 
On  pricking  this,  serum  loaded  with  the  bacilli  will 
exude.  The  bacilli  are  not  found  in  ulcers  or  sores 
in  purely  nerve  leprosy,  as  in  that  form  they 
are  present  in  the  nerve  sheaths  and  the  ulcer  or 
necrosis  is  not  due  to  the  breaking  down  of  a 
leprous  granuloma,  or  of  tissues  infiltrated  with  the 
organisms. 

One  of  the  most  constant  natural  discharges  to 
contain  the  bacilli  is  the  mucus  discharged  from 
the  nose.  It  is  also  one  of  the  earliest  manifesta- 
tations  in  many  cases,  including  some  of  nerve  or 
anaesthetic  leprosy.  By  some  it  is  believed  that 
the  earliest  and  most  constant  lesion  of  leprosy  of 
all  forms  is  a  deposit  in  or  below  the  nasal  mucous 
membrane. 

Various  differences  in  size,  staining  reaction,  &c, 
between  lepra  and  tubercle  bacilli  have  been  de- 
scribed, but  they  are  not  sufficiently  marked  or 
constant  to  be  of  diagnostic  value.  The  most  im- 
portant diagnostic  point  in  films  of  mucus  is 
the  aggregation  of  the  bacilli  into  small,  dense 
clumps  in  leprosy,  in  many  cases  still  retaining  the 
outline  of  the  cell  in  which  they  grew.  In  sections 
of  skin  the  extraordinary  profusion  of  the  organisms, 
as  well  as  the  aggregation  into  compact  masses,  is 
characteristic  of  lepra  "bacillus  as  opposed  to  human 
tubercle.  In  some  of  the  lesions  of  avian  tubercle 
a  similar  grouping  may  be  found,  whilst  in  internal 
organs  in  man  there  may  be  diffuse  growth  of  the 
lepra  bacilli. 


_j.OO  SMEGMA 

Smegma  Bacilli. —  This  group  probably  includes 
several  species.  In  most  specimens  of  smegma  the 
organisms,  though  truly  acid  fast,  are  decolourized 
by  alcohol,  that  is,  they  are  not  alcohol  fast.  Some 
varieties,  however,  do  not  lose  their  stain  in  alcohol, 
and  are  like  the  tubercle,  both  acid  and  alcohol 
fast.  This  organism  has  been  the  cause  of  frequent 
mistakes  in  diagnosis,  as  urine  easily  becomes  con- 
taminated with  this  bacillus,  and  it  may  readily  be 
mistaken  for  that  of  tubercle  and  a  diagnosis  of 
urinary  or  renal  tuberculosis  given.  In  the  majority 
of  cases  the  use  of  alcohol,  as  well  as  of  acid,  will 
prevent  this  mistake,  but  as  some  specimens  of  the 
smegma,  including  Lustgarten's  so-called  syphilis 
bacillus,  are  also  alcohol  fast,  the  possibility  of  the 
confusion  should  be  avoided  by  using  the  catheter. 

The    fourth   group,   of    which    the  Timothy  grass 
bacillus,   or  B.  pJilei,   is  taken  as  the  type,  are    the 
only  organisms  of   this    group    which  grow  readily 
on  almost  any  medium.     They  are  found  on  several 
species  of  grass  used  as  fodder,  and  may  be  found 
in  enormous   numbers  m  the  faeces  of  cattle.     As  a 
consequence    they    are    often    found    in    milk    and 
products,   such  as  butter  and   cheese,   derived  from 
milk.      Several    varieties   or    species    have    been  de- 
scribed.     This   group    is    of    economic    importance, 
as  cattle   have  been  condemned  as  tuberculous,  on 
the  grounds  that  acid-fast  bacilli  were  found   in  the 
stools  and  milk  ;  butter  and  cheese  have  also  been 
condemned   on   this   insufficient  reason.     These  or- 
ganisms are  not  pathogenic. 
(6)  Growths  on  Artificial  Media. — With  organisms  that 
can  be  cultivated,  the  growths  on  artificial  media  (nutrient 
broth,  gelatine,  or  agar),  differ  in   some  cases  sufficiently 
to  be  of  diagnostic  value,  and  in  any  case  the  character 
of    the  growth  is  one  of    the  properties  of  an  organism 
that  requires  description. 

Cultures  may  be  made  on  plates  as  in  the  separation 


CHARACTERS   OF   CULTURES  -\OJ 

of  different  organisms,  or,  and  more  conveniently,  in 
tubes.  The  growths  in  fluid  media  are  made  by  taking 
on  a  sterilized  platinum  loop  a  portion  of  the  culture 
and  inoculating  the  tube.  The  nature  and  character 
of  the  naked-eye  appearance  in  the  broth  at  varying 
periods  should  be  described.  The  temperature  at  which 
the  cultivation  is  made  must  also  be  noted  where  incu- 
bators are  available.  Blood  heat,  370  C.  and  210  C.  are 
the  most  convenient,  and  terms  like  "room  temperature" 
should  be  avoided.  In  many  tropical  countries  the 
temperature  of  the  air  will  range  from  250  to  300  C,  and 
satisfactory  growths  of  the  more  important  organisms 
can  be  obtained.  In  a  description  of  the  growths 
at  these  temperatures  a  result  intermediate  between 
those  at  incubator  temperatures  will  be  obtained.  All 
cultivations  must  be  carried  on  in  the  dark. 

The  points  to  be  observed  in  a  broth  culture  are  the 
surface,  whether  covered  with  or  free  from  a  film  or 
pellicle.  In  the  body  of  the  fluid  note  if  the  fluid  is 
turbid  and  the  degree  of  turbidity,  if  not  turbid  whether 
quite  clear  or  with  floating  particles ;  the  presence  or 
absence  of  a  precipitate  and,  if  one  be  present,  whether 
it  is  composed  of  a  uniform  fine  deposit  or  if  in 
separate  masses.  Any  change  in  colour,  and  bubbles 
from  formation  of  gas,  must  be  further  noted. 

On  solid  media  the  growths  may  be  observed  on  plates 
or  in  tubes.  In  tubes  the  growths  can  be  seen  on  sloped 
cultures  made  by  drawing  the  inoculated  platinum  loop 
over  the  surface  of  the  medium.  This  sloped  medium  is 
obtained  by  placing  the  tube,  whilst  the  medium  is  still 
liquid,  in  a  sloped  position,  and  allowing  it  to  set,  or 
stab  cultures  may  be  used.  In  these  the  medium  is 
allowed  to  set  with  the  tubes  vertical.  An  inoculated 
wire,  not  a  loop,  is  plunged  steadily  into  the  depths  of 
the  medium  and  withdrawn  without  splitting  the  medium. 

The  appearance  of  the  separate  colonies  is  most  im- 
portant.     There  are  great  diversities   in  the  appearance 
of    growths   on  solid   media,  and  an  accurate  series  of 
26 


402  CHARACTERS 

defined  terms  for  descriptive  purposes  is  much  needed. 
Such  a  series  of  descriptive  terms  has  been  drawn  up 
by  Chester,  but  many  of  the  terms  will  probably  not  be- 
generally  accepted  and  they  are  used  at  present  by  few- 
bacteriologists. 

The  observer  should  note  and  describe  the  size  of 
the  individual  colonies,  their  shape,  the  character  of 
the  edge,  their  elevation,  whether  raised  or  depressed, 
and  give  a  detailed  account  of  the  character  of  the 
surface.  The  macroscopic  appearances  of  the  colony  : 
if  transparent,  whether  highly  retractile  or  not,  and  if 
not  clear  whether  opalescent,  finely  or  coarsely  granular, 
or  irregularly  blotchy.  Any  colouration,  either  of  the 
colony  itself  or  the  surrounding  medium,  must  be  noted. 
In  some  organisms  the  different  colonies  remain  distinct 
even  when  in  contact,  whilst  with  other  organisms  adjoin- 
ing colonies  readily  grow  together  or  become  confluent. 
In  growths  on  gelatine  the  presence  or  absence  of  signs 
of  liquefaction  in  the  surrounding  medium  is  a  point 
of  the  first  importance. 

In  stab  cultures  any  surface  growth  must  be  noted,  as 
well  as  the  growth  in  the  line  of  puncture.  It  may  be 
uniform,  finely  or  coarsely  granular,  composed  of 
numerous  fine  or  coarse  colonies  which  remain  discrete 
and  are  not  confluent,  or  of  large  masses.  Extension 
into  the  gelatine  in  the  neighbourhood  of  the  puncture 
may  take  place,  and  the  character  of  these  extensions, 
whether  as  knots  or  as  fine  filaments,  or  in  an  irregular, 
arborescent  manner,  is  worthy  of  attention. 

If  liquefaction,  in  a  gelatine  medium,  has  taken  place 
it  will  be  well  shown.  It  is  most  abundant  in  the  upper 
part  of  the  line  of  puncture  when  the  organism  requires 
oxygen,  but  with  organisms  that  grow  best  in  the  absence 
of  air  will  be  more  conspicuous  in  the  depths. 

Air  bubbles  along  the  line  of  puncture,  indicating 
formation  of  gas  and  any  colouration  of  the  growth  or 
of  the  medium,  must  be  noted. 

The  amount  of  growth  that  takes  place  in  a  given  time 


CONDITIONS   AFFECTING   GROWTH  403 

as  compared  with  other  organisms,  or  similarly  the 
relative  amount  of  liquefaction,  gas  formation,  &c,  in 
the  time,  is  an  aid  in  distinguishing  allied  or  similar 
organisms,  though  liable  to  be  modified  with  different 
strains  of  the  same  organism. 

There  is  no  cultural  characteristic  that  cannot  be 
modified  by  frequent  subculture,  culture  under  different 
conditions  of  temperature,  reaction  of  medium,  and  other 
influences.  The  cultures  of  some  organisms  vary  more 
than  those  of  others.  The  information  gained  as  to  the 
character  of  growths,  though  of  considerable  value,  has 
to  be  considered  with  other  properties  of  the  organisms. 

Cultures  on  milk,  potatoes,  &c,  are  often  of  more 
diagnostic  value  for  special  organisms. 

(7)  Conditions  Affecting  the  Growth  of  Organisms. — One 
of  these,  the  effect  of  oxygen,  is  of  special  practical  value. 

Some  organisms  will  only  grow  in  presence  of  oxygen  ; 
such  organisms  are  strictly  aerobic.  Others  will  not  grow 
at  all  in  the  presence  of  oxygen,  these  are  said  to  be 
strictly  anaerobic.  The  largest  number  of  bacteria  are 
intermediate  between  the  two  and  are  termed  facultative 
anaerobes. 

Aerobic  organisms  grow  readily  under  the  ordinary 
conditions,  as  even  in  stab  cultures  there  is  usually 
sufficient  oxygen  present  for  the  commencement  of 
growth. 

Anaerobic  organisms  are  most  easily  grown  in  stab 
cultures  of  glucose-agar  or  gelatine,  or  in  glucose  formate 
agar  made  by  adding  '02  per  cent.  sod.  formate  to  glucose 
agar  (Kitisato).  The  tubes  must  have  been  freshly 
boiled  to  expel  air  from  the  medium,  and  the  stab  should 
be  made  with  a  fine  needle  so  as  to  carry  down  as  little 
air  as  possible.  After  the  needle  is  withdrawn  the  upper 
part  of  the  medium  should  be  heated  so  as  to  melt  it  and 
seal  the  opening  made  by  the  needle. 

It  must  be  remembered  that  though  the  growth  of 
anaerobic  organisms  does  not  take  place  in  presence 
of  air,   the   organisms,  and  particularly  the  spores,  may 


-1(>4 


CHEMICAL    PRODUCTS 


retain   their  vitality  and  grow   it"   transplanted    into   more 
favourable  conditions. 

(8)  Chemical  Products  of  Organisms. — These  vary  both 
with  the  nature  of  the  organism  and  the  character  of  the 
medium.  Gas  formation  is  one  of  the  easiest  to  deter- 
mine, but  it  is  also  necessary  to  have  in  the  medium 
some  substance  from  which  the  gas  can  be  formed.  The 
sugars  are  valuable  for  this  purpose,  and  it  will  be  found 
that  whilst  one  organism  will  form  gas  from  either 
glucose  or  lactose  another  will  form  gas  only  from 
glucose.  Another  manifestation  of  chemical  activity  is 
the  formation  of  acid  or  alkali.  Formation  of  acids  and 
gases  are  of   particular    importance,    as    so  many  of  the 


Fig.  157. 

organisms  found  in  the  intestine  either  form  gas  and  acid 
from  glucose  or  form  acid  only. 

Formation  of  Gas. — The  production  of  gas  can  be 
shown  in  most  stab  preparations  as  bubbles  of  gas  form 
along  the  needle  track.  It  is  better  shown  by  melting 
the  gelatine,  or,  better,  glucose  gelatine,  and  rotating  the 
tube  alternately  in  opposite  directions  after  inoculation. 
Such  a  preparation  is  known  as  a  "  shake  culture,"  and 
after  it  has  set  and  grown,  bubbles  of  gas  will  be  formed 
all  through  the  medium. 

A  better  method  that  can  be  used  with  fluid  media  is 
to  place  in  the  medium  a  small  inverted  tube,  Durham's 
tube.  During  sterilization  of  the  medium  the  gas  will 
be  expelled  from  this  small  tube,  which  will  be  completely 
filled  with  the  fluid  medium.     If  the  tube  be  inoculated 


GAS   AND   ACID  405 

with  an  organism  that  forms  gas  from  the  medium  the 
gas  formed  in  the  small  inverted  tube  will  accumulate 
in  it  and  cause  it  to  float  (fig.  157.) 

Gas  formation  from  glucose  is  one  of  the  characteristics 
of  some  of  the  commoner  intestinal  bacteria. 

FORMATION  OF  Acid. — Acid  formation  can  be  shown 
by  using  a  neutral  or  slightly  alkaline  medium  coloured 
with  litmus  ;  the  formation  of  acid  is  shown  by  the 
change  in  colour  of  the  litmus. 

Most,  of  the  intestinal  bacteria  form  acid  readily.  An 
ingenious  application  of  these  properties  of  the  intestinal 
organisms  is  that  of  MacConkey  for  the  detection  of 
faecal  contamination  of  water,  milk,  and  other  substances. 
He  uses  Durham's  tubes  and  employs  a  medium  con- 
taining bile  salts.  Bile  salts  inhibit  the  growth  of  many 
organisms,  but  are  favourable  to  the  growth  of  intestinal 
bacteria. 

The  medium  he  employs  is  composed  of :  Peptone,  2  ; 
salt,  *5  ;  sodium  taurocholate,  "5  ;  water,  100  ;  to  which 
is  added  glucose  or  lactose  in  the  proportion  of  "5  per 
cent.  The  medium  is  neutral  and  is  coloured  with  neutral 
litmus.  A  Durham's  tube  is  placed  in  the  test  tube 
containing  the  medium  and  during  the  three  sterilizations 
required  will  be  filled  with  the  medium. 

A  measured  amount  of  the  water,  &c,  to  be  tested 
is  added  and  the  tube  incubated,  preferably  at  420  C,  for 
twenty-four  hours.  Organisms  which  in  this  medium 
produce  acid  and  gas  may  be  suspected  to  be  possible 
inhabitants  of  the  intestinal  tract. 

Other  pathogenic  and  non-pathogenic  intestinal  or- 
ganisms form  acid  only.  Of  organisms  other  than  these 
many  will  not  grow  in  the  medium  at  all,  or  if  they  do 
grow  form  neither  acid  nor  gas. 

If,  therefore,  neither  acid  nor  gas  is  formed,  the  evi- 
dence is  strong  that  there  is  no  living  faecal  contamina- 
tion, and  no  contamination  with  the  commonest  of  the 
intestinal  organisms,  B.  coli  communis.  If  acid  alone  is 
formed  it  is  doubtful  whether  there  is  such  contamination, 


40O  IXDOL   FORMATION 

as  />'.  coli  communis  must  be  absent.  If  acid  and  gas  are 
both  formed  there  is  strong  probability  that  the  water, 
&c,  is  contaminated  with  organisms  that  are  inhabitants 
of  the  intestinal  tract. 

Indol  Formation  is  another  important  chemical  pro- 
duct of  some  bacteria.  A  simple  medium  is  required, 
and  plain  peptone  water  made  by  boiling  10  grm.  of 
peptone  and  5  grm.  of  salt  in  a  litre  of  water  is  usually 
employed.     This  should  be  filtered  and  sterilized  as  usual. 

The  tube  of  this  medium  should  be  inoculated  with 
the  organism  to  be  examined  and  incubated  for  at  least 
twenty-four  hours.  Other  tubes  inoculated  at  the  same 
time  are  incubated  for  longer  periods. 

To  this  culture  a  little  pure  sulphuric  acid  is  added  ; 
a  red  colour  develops  in  a  few  minutes  if  indol  and  a 
nitrite  have  been  formed.  If  the  colour  remains  unal- 
tered, three  or  four  drops  of  a  '05  solution  of  sodium 
nitrite  should  be  added  to  the  mixture,  and  if  a  red 
colour  now  develops  indol  alone  has  been  formed. 
Yellow-fuming  nitric  acid,  which  contains  traces  of  nitrous 
acid,  may  be  satisfactorily  used  instead  of  sod.  nitrite. 

Amongst  other  chemical  products  are  ammonia,  alcohol, 
phenol,  sulphuretted  hydrogen,  and  the  substances  which 
cause  curdling  of  milk. 

Effects  ox  certain  Aniline  Colouring  Matters. 

Neutral-red  Agar. — This  medium  consists  of  ordinary 
agar,  to  each  100  c.c.  of  which  "3  grm.  of  glucose  and 
1  c.c.  of  a  saturated  aqueous  solution  of  neutral-red 
have  been  added  before  the  medium  is  poured  into  tubes. 

In  this  medium  B.  typhosus  and  B.  dysenteries  grow- 
without  changing  it,  while  B.  coli  communis  and  the 
paratyphoid  bacilli,  in  twenty-four  to  forty-eight  hours 
decolourise  the  medium  and  produce  a  greenish  fluores- 
cence, forming  gas  at  the  same  time.  Stab  cultures  or 
shake  cultures  may  be  employed. 

Drigalski-Conradi's  Medium. — To  prepare  this  medium, 
2,000    c.c.    of    3     per    cent,    nutrient    agar    are    treated 


SERUM    REACTIONS  407 

with  20  grm.  nutrose,  then  with  a  solution  of  30 
grm.  of  lactose  in  260  c.c.  litmus  solution.  The  pro- 
cedure is  as  follows  :  The  litmus  solution  is  boiled  for 
ten  minutes  in  the  steam  sterilizer,  the  lactose  added,  and 
the  mixture  again  boiled  for  ten  minutes.  The  litmus 
lactose  solution  is  cooled  to  40°to  500  C,  and  the  nutrose 
agar  cooled  to  700  C.  is  added  to  it.  The  mixture  is 
rendered  alkaline  with  hot  10  per  cent,  soda  solution  to 
the  extent  that,  on  shaking,  the  froth  formed  is  distinctly 
blue  after  a  few  minutes'  standing.  Finally,  20  c.c.  of 
o- 1  per  cent,  freshly  prepared  solution  of  crystal  violet  are 
added,  and  the  medium  sterilized  in  the  usual  way — it 
should  be  bluish  violet  when  solidified. 

On  this  medium  B.  typhosus  produces  small  transparent 
colonies,  while  B.  coll  com  munis  produces  larger  colonies, 
brilliant  red  and  non-transparent. 

(9)  Reaction  of  Oigauisms  with  various  Blood  Sera. — 
Certain  pathogenic  organisms  effect  a  change  in  the 
blood  serum  of  persons  infected  with  these  organisms, 
so  that  the  serum  contains  substances  which,  when  mixed 
with  a  living  culture  of  the  organism,  cause  loss  of 
motility  of  the  bacteria,  and  also  cause  them  to  aggregate 
in  little  clumps  or  masses.  This  aggregation  is  called 
agglutination,  and  the  serum  which  causes  this  agglu- 
tination is  said  to  contain  agglutinins. 

The  application  of  the  test  is  simple.  The  blood 
serum,  free  from  red  corpuscles,  is  obtained,  as  has  been 
already  described,  and  diluted  with  sterile  broth  to  a 
known  extent,  ten,  twenty,  thirty,  or  more  times.  Some- 
times the  blood  is  sent  in  capillary  tubes  or  the  serum 
has  not  well  separated.  In  such  cases,  to  obtain  clear 
serum  it  is  necessary  to  use  the  centrifuge  (tig.  158). 
This  diluted  serum  is  mixed  with  an  equal  volume  of  a 
living  active  culture  of  the  organism  to  be  tested  and  the 
mixture  examined  as  a  hanging-drop  preparation.  Loss 
of  motion  of  the  organisms  and  agglutination  should 
take  place  within  a  certain  time  limit  and  a  control  made 
by  using  the  same  dilution  of  normal  blood  serum  with 


408 


SERUM    REACTIONS 


more  of  the  same  culture.  This  action  of  the  serum  on 
the  organisms  is  specific  and  affords  a  means  of  proving 
the  correlation  of  the  organism  and  disease. 

It  is  true  that  strong  undiluted  normal  serum  will  cause 
in  some  cases  a  similar  agglutination,  but  not  with  the 
great  dilutions  which  will  act  in  serum  from  a  person 
with  disease. 


Fig.   158. — Centrifuge. 


The  converse  of  this  test  is  to  use  a  culture  of  an 
organism  to  test  the  serum  reaction  of  a  patient  suspected 
to  be  suffering  from  the  disease  which  the  organism  can 
cause.  Typhoid  and  Malta  fever  are  the  diseases  in  which 
the  reaction  is  most  decisive.      This  application  of   the 


SERUM    REACTIONS  409 

principle  is  known  as  the  agglutination  test,  or  the 
Griinbaum-Widal  or  Widal  reaction. 

In  the  application  of  the  hanging-drop  method  the 
serum  may  be  diluted  in  Wright's  tubes  or  by  a  number 
of  loopfuls  of  broth  being  added  to  a  loopful  of  serum 
and  well  mixed.  To  obtain  a  dilution  of  1  in  20,  one 
loopful  of  serum  is  mixed  with  nine  loopfuls  of  broth, 
and  of  this  mixture  one  or  two  loopfuls  are  mixed  with 
one  or  two  loopfuls  of  an  active  culture. 

If  higher  dilutions  of  the  serum  only,  say  1  in  100,  are 
required,  it  would  be  inconvenient  and  tedious  to  mix 
one  loopful  with  ninety-nine  of  broth.  It  is  easier  to 
make  a  dilution  of  1  in  10  and  dilute  one  loopful  of 
this  dilution  with  nine  of  broth,  which  gives  the  same 
dilution  more  quickly. 

The  gradual  loss  of  motility  and  aggregation  of  the 
organisms  can  be  watched  under  the  microscope  in  the 
hanging-drop  preparation. 

Many  observers  prefer  the  macroscopic  demonstrations 
of  the  same  effect.  This  is  done  by  aspirating  into  a 
tube  a  mixture  of  serum  diluted  to  the  required  extent 
with  broth  mixed  with  an  equal  quantity  of  active  broth 
culture.  The  mixture  of  culture  and  diluted  serum  is 
drawn  up  into  a  narrow  tube  and  placed  vertically  in 
the  incubator.  The  organisms  will  lose  their  motility 
and  aggregate  into  a  mass  and  fall  to  the  bottom  of  the 
fluid,  leaving  the  superjacent  fluid  clear  and  free  from 
turbidity,  in  marked  contrast  to  the  control,  which  will 
still  remain  turbid.  This  method  is  known  as  the  "  sedi- 
mentation test." 

There  are  many  fallacies  which  may  occur  in  con- 
nection with  these  tests.  The  culture  must  be  an  active 
one  and  recently  made.  A  control  with  serum  of  normal 
blood  must  be  made  and  the  dilution  must  be  sufficient. 

Some  strains  of  the  organisms  agglutinate  more  readily 
than  others,  and  even  with  diluted  serum  of  normal 
blood,  agglutination  may  take  place  if  the  organisms  are 
grown  on  unsuitable  media,  or  if  the  cultures  are  too  old. 


410  PATHOGENIC   PROPERTIES 

The  change  in  the  serum  may  be  a  persistent  one,  so 
that  a  positive  reaction  in  the  case  of  a  person  who  has 
had  a  previous  attack  of  typhoid  or  Malta  fever  gives  no 
information  as  to  his  present  condition. 

(10)  Pathogenic  Properties. — The  pathogenic  properties 
of  an  organism  are  shown  by  the  effect  of  inoculating  a 
susceptible  animal  with  the  organism  in  pure  culture  if 
possible.  Where  that  is  not  possible,  with  fluid  contain- 
ing as  few  other  organisms  as  possible. 

In  some  instances,  as  in  tuberculosis,  the  similarity  of 
the  lesions  produced  by  a  similar  organism  in  animals 
naturally  indicated  that  cultures  of  tubercle  bacilli  should 
be  tried  on  other  animals.  In  others  a  series  of  animals 
had  to  be  tried  before  a  susceptible  host  was  found. 
Rats,  guinea-pigs  and  rabbits  are  the  animals  most  com- 
monly used,  but  in  other  cases  monkeys,  dogs,  cattle 
and  horses  have  had  to  be  employed.  No  such  experi- 
ments can  be  made  under  the  Vivisection  Acts  without 
a  licence,  and  in  any  case  there  are  so  many  difficulties 
and  fallacies  that  without  a  thorough  study  of  these  and 
of  the  methods  employed  the  results  obtained  would  be 
valueless. 

Material  used  for  injection  may  be  :  — 

(i)   Pure  cultures  of  an  organism. 

(2)  Products  of  bacteria  in  solution  such  as  toxins. 

(3)  Fluid  excretions,  secretions  and  portions  of  diseased 
tissues. 

(4)  Blood. 

The  injections  are  usually  made  with  strict  antiseptic 
precautions  into  the  subcutaneous  cellular  tissues.  With 
fluid  cultures  there  is  no  special  difficulty.  Cultures  on 
solid  media  require  to  be  emulsified  with  sterile  saline 
solution.  Solid  tissues,  portions  of  spleen,  &c,  should 
be  rubbed  up  in  a  sterile  glass  mortar  with  a  little  sterile 
broth  and  then  injected.  Occasionally  a  small  mass  of 
solid  tissue  is  inserted  into  an  aseptic  pocket  made  under 
the  skin  for  the  purpose,  and  the  wound  closed  by  a 
sealed  dressing  such  as  gauze  and  collodion. 


INJECTIONS  4II 

In  other  cases  the  injection  is  intramuscular  or  intra- 
peritoneal. In  intraperitoneal  injections  of  small  animals 
a  big  fold  should  be  taken  up  between  the  finger  and 
thumb,  care  being  taken  to  include  the  whole  thickness 
of  the  abdominal  wall  and  to  see  that  no  intestine  is 
included.  Whilst  still  held  this  fold  should  be  transfixed 
with  the  needle  of  the  hypodermic  syringe  containing 
the  substance  to  be  injected  so  that  the  point  just  pro- 
trudes. The  finger  and  thumb  are  now  removed,  the 
abdominal  wall  will  flatten  out  and  the  point  of  the 
needle  will  be  in  the  abdominal  cavity.  In  this  way 
the  injection  can  be  made  without  any  risk  of  injuring 
the  intestines. 

The  results  of  inoculations  are  not  always  conclusive. 
The  resulting  disease,  even  in  a  susceptible  animal,  may 
show  very  little  resemblance  to  the  disease  caused  by 
the  same  organism  in  man  or  other  animals. 

An  organism  that  is  pathogenic  may  by  successive 
cultures  lose  its  virulence,  and  become  with  some 
animals  non-pathogenic,  whilst  on  the  other  hand,  if 
passed  through  a  series  of  animals,  the  virulence  may 
be  increased. 

Koch's  postulates  are  : — 

(1)  The  organisms  must  be  found  in  the  tissues,  fluids 
or  organs  of  the  animal  affected  with  the  disease. 

(2)  The  organism  must  be  isolated  and  cultivated 
outside  the  body  on  suitable  media  for  successive 
generations. 

(3)  The  isolated  and  cultivated  organism  on  inoculation 
into  a  suitable  animal  should  reproduce  the  disease. 

(4)  The  same  organism  must  be  recoverable  from  the 
inoculated  animal. 

These  in  the  main  are  still  considered  sound,  though 
not  practicable  for  all  organisms,  as  some  cannot  be 
cultivated  :  for  others  no  susceptible  animal  is  yet 
known,  and  in  some  of  the  lower  animals,  though  a 
disease  is  produced  by  the  injections,  it  bears  little  or  no 
resemblance  to  the  human  disease  under  investigation. 


412  MYCETOMA 

Streptothrix  madurce. — This  organism  is  the  cause  <>! 
a  disease  of  special  importance  in  the  Tropics — madura 
foot.  It  occurs  in  India,  Straits  Settlements,  East  Africa, 
British  Guiana,  Cyprus,  and  Cuba,  and  is  probably  to  be 
met  with  all  through  the  Tropics. 

Clinically  it  is  a  chronic  disease  which  causes  much 
swelling  of  a  firm  fibrous  nature  and  destruction  of  the 
tissues,  with  the  formation  of  sinuses  discharging  watery 
or  oily-looking  fluid.  In  some  cases  in  this  discharge, 
white,  black,  or  pink  granules,  visible  to  the  naked  eye, 
are  found,  and  these  granules  are  masses  of  branching 
filaments  with  mycelial  arrangement.  In  other  cases  the 
granules  are  very  rare,  and  only  the  branching  filaments 
of  the  mycelium  are  found  ;  in  still  other  cases  these 
may  also  be  absent,  though  in  sections  of  the  tissue  the 
mycelial  clumps  are  to  be  seen. 

The  organism  in  tissue  is  characterized  by  the  dense 
clumps  of  mycelium  formed  of  the  branching  fila- 
ments of  the  streptothrix.  The  ends  of  the  filaments 
at  the  edges  of  these  masses  degenerate  and  become 
swollen,  forming  the  so-called  clubs. 

The  organism  will  grow  on  any  of  the  ordinary  media, 
forming  limpet-shaped  masses. 

In  general  character  the  mycetoma  resembles  the 
streptothrix  of  actinomycosis,  but  it  does  not  stain  by 
Gram  in  sections,  does  not  liquefy  gelatine,  and  the 
"clubs"  are  rounder.  The  clumps  take  any  of  the 
ordinary  basic  stains,  including  hematoxylin,  and  either 
this  stain  or  carbolthionin  is  to  be  recommended  to  show 
the  growth  in  sections. 

This  streptothrix  sets  up  changes  in  the  tissue,  so 
that  the  growths  are  surrounded  by  a  mass  of  newly 
formed  tissue  of  the  granulomatous  type.  At  the  peri- 
phery of  the  granulomatous  mass  is  much  badly  formed 
fibrous  tissue,  and  the  centre  is  often  broken  down 
It  is  in  the  breaking  down  of  this  granulomatous  tissue 
that  the  mycelium  clumps  are  liberated  and  are  dis- 
charged with  the  fluids  from  the  sinuses. 


water  analysis  413 

Bacteriological  Examination  of  Water. 

In  the  bacteriological  analysis  of  water  we  determine 
(1)  the  total  number  of  organisms  of  all  kinds  in  a 
given  quantity  of  the  sample  ;  (2)  the  presence  or  absence 
of  B.  coli  communis;  (3)  the  detection  of  definite  patho- 
genic organisms,  such  as  the  B.  typhosus,  Koch's  comma 
bacillus,  &c. 

The  enumeration  of  the  number  of  organisms  irre- 
spective of  the  kind  is  of  value  when  we  wish  to  deter- 
mine the  efficiency  of  filter  beds,  effects  of  sedimentation, 
storage,  &c. 

The  detection  of  B.  coli  communis  is  the  main  object 
of  any  bacteriological  examination.  Not  that  B.  coli 
per  sc  is  to  be  regarded  as  a  definite  pathogenic  organism, 
but,  occurring  as  it  does  in  all  dejecta,  its  presence  is 
regarded  as  an  indicator  of  sewage  contamination. 
Where  sewage  contamination  is  shown  to  have  occurred, 
it  is  quite  possible  that  other  and  more  deadly  organisms 
occurring  in  the  intestinal  tract,  such  as  B.  typhosus  and 
Koch's  comma  bacillus,  may  also  have  gained  access  to 
the  water  under  consideration.  The  B.  typhosus  has  but 
rarely  been  isolated  from  a  water  supply,  though  Koch's 
comma  bacillus  can  readily  be  isolated  from  water  con- 
taining these  organisms. 

Perhaps  the  simplest  and  best  method  of  proceeding  to 
make  a  bacteriological  examination  of  any  given  water  is 
that  recommended  by  Savage,  which,  with  slight  modifi- 
cations, is  as  follows  : — 

Collection. — Not  less  than  2  oz.  to  be  collected  in  a 
sterile  glass-stoppered  bottle.  When  taking  a  sample 
from  a  tap,  allow  the  water  to  run  to  waste  for  some 
live  minutes  before  collecting  the  specimen  ;  when  from 
a  pond  or  river,  the  sample  should  be  collected  well 
away  from  the  bank.  If  the  water  of  a  well  is  to  be 
examined,  the  sample  should  not  be  taken  from  the 
surface  of  the  water,  but  from  a  point  about  a  foot 
deeper.  It  is  best  to  pack  all  samples  in  ice,  and  transmit 
at  once  to  the  laboratory. 


414  WATER   ANALYSIS 

Inoculations. — Everything  must  be  in  readiness  before 
the  examination  is  started,  i.e.,  gelatine  and  agar  tubes 
melted,  and  at  a  suitable  temperature,  other  media  tubes 
ready,  sterile  pipettes  and  Petri  dishes  at  hand.  Use  a 
1  c.c.  pipette  graduated  in  TT0  c.c.  Mix  the  sample 
thoroughly.  Add  o"2,  0*3,  and  0-5  c.c.  respectively  to 
three  gelatine  tubes,  and  label  with  a  grease  pencil.  Add 
o*i  and  ro  c.c.  respectively  to  two  agar  tubes  ;  label  and 
replace  at  once  in  the  hot-water  bath.  Add  ro  cc.  to  a 
tube  of  bile  salt  broth.  The  effect  of  this  medium  is  to 
inhibit  the  growth  of  organisms  other  than  those  which 
flourish  in  the  intestinal  tract.  Add  10  c.c.  to  a  tube  of 
double-strength  bile  salt  broth.  All  tubes  of  bile  salt  broth 
are  provided  with  Durham's  tubes.  Add  *i  c.c.  to  a  tube 
of  glucose  neutral  red  broth  provided  with  a  Durham's  tube. 
Add  1  c.c.  to  a  tube  of  glucose  neutral  red  broth  provided 
with  a  Durham's  tube.  Add  10  c.c.  to  a  tube  of  glucose 
neutral  red  broth  double  strength,  and  provided  with  a 
Durham's  tube.  To  the  water  remaining  in  the  bottle 
add  the  contents  (about  10  c.c.)  of  a  tube  of  four  times 
strength  neutral  red  broth.     Replace  the  stopper. 

The  gelatine  and  agar  tubes  are  now  poured  into  Petri 
dishes  after  thorough  admixture  of  water  and  medium  has 
been  made.  The  Petri  dishes  are  labelled,  and  their 
contents  rapidly  solidified. 

The  agar  plates  are  incubated  at  370  C,  upside  down, 
and  the  gelatine  plates  at  220  C  ,  but  not  reversed. 

The  bile  salt  and  neutral  red  broth  tubes  are  labelled 
and  incubated  at  370  C. 

Examination  of  Plates  and  Tubes. 

Plates. — Gelatine  and  agar  plates  should  be  counted  at 
the  end  of  twenty-four,  forty-eight,  and  seventy-two 
hours  ;  but  in  all  cases  the  plates  should  be  inspected 
earlier,  in  order  that  the  count  may  be  made  at  once 
should  liquefaction  render  this  necessary.  To  count  the 
colonies,  it  is  best  to  count  against  a  dark  background, 
and  with  a  brush  place  a  dot  of  Indian  ink  over  each 
counted    colony.       In    this   way,    as   the    older,    already 


WATER   ANALYSIS  415 

counted  colonies  are  marked,  the  number  of  new  colonies 
that  become  visible  each  day  can  be  noted. 

To  facilitate  counting,  divide  up  the  area  of  the  plate 
with  lines  on  the  back  made  with  a  grease  pencil.  All 
the  colonies  on  the  plate  should  be  counted  ;  but  if  they 
are  very  numerous,  and  an  approximate  estimate  only  is 
possible,  then  some  mechanical  aid  such  as  Pake's  disc 
may  be  used,  a  few  segments  being  counted,  and  the  total 
number  deduced. 

Tubes. — For  B.  coll. — If  the  1  c.c.  and  10  cc.  bile  salt 
and  neutral  red  broth  tubes  show  no  gas  or  reaction 
after  forty-eight  hours,  it  can  be  assumed  that  B.  coli  is 
absent  in  these  amounts.  Then  in  every  case  plate  out 
from  the  broth  and  water  in  the  sample  water. 

If  gas  is  formed  in  any  of  these  tubes,  use  the  one 
showing  gas  in  the  tube  with  the  least  quantity  of  added 
water  for  inoculating  plates  of  solid  media.  For  the 
actual  isolation,  it  is  sufficient  to  add  one  platinum 
loopful  of  the  medium  to  a  wide  tube,  containing  sterile 
water,  and  to  distribute  a  little  of  this  over  plates  of  solid 
media  suitable  for  the  purpose.  Such  media  are  lactose 
litmus  agar,  Conradi  medium,  and  neutral  red  bile  salt 
agar. 

For  spreading  the  diluted  broth  over  the  plates  of  solid 
media,  a  common  method  is  to  employ  a  glass  rod  bent 
at  right  angles  near  one  end.  The  diluted  broth  is  placed 
on  the  solid  medium  in  the  plate,  and  distributed  by 
means  of  the  sterilized  glass  rod. 

By  the  next  day,  if  the  plates  have  been  incubated 
at  370  C,  the  colonies  will  have  developed  sufficiently 
for  examination  and  subcultivations,  of  which  at  least 
three  should  be  made. 

Having  now  isolated  the  coli-like  organism  in  pure 
culture,  it  should  be  further  tested  on  various  media  to 
see  that  it  conforms  absolutely  to  the  reactions  produced 
by  an  undoubted  B.  coll. 

A  coined  word,  "  flaginac,"  is  often  used  to  express  the 
results  of  subcultural  tests  of  coli-like  organisms.  The 
word  is  made  up  as  follows  : — 


416  [NTERPRETATION   OF   RESULTS 

//.  indicates  greenish   fluorescence  in   neutral   red- 
broth  cultures. 

ag.   indicates    acid    and    gas    in     lactose    peptone 

cultures. 

in.  indicates  indol  formation  in  broth  cultures. 
ac.  indicates  acidity  and  clotting  of  litmus  milk. 
Examination  for  Koch's  Com  ma  Bacillus. — About  a  litre 
of  the  water  is  placed  in  twelve  large  sterile  Erlenmeyer 
flasks,  90  c.c.  in  each.  To  each  is  added  10  c.c.  of  a 
sterile  solution,  consisting  of  10  per  cent,  peptone  and 
5  per  cent,  sodium  chloride.  The  flasks  are  then  incu- 
bated at  37°  C.  After  eighteen  hours'  incubation  micro- 
scopic preparations  and  examinations  in  hanging  drop 
are  made  from  the  surface  of  each  flask.  The  medium  is 
one  in  which  the  cholera  spirillum  grows  very  rapidly, 
and,  if  present,  it  shows  itself  in  the  very  thin  pellicle  on 
the  surface  of  the  liquid,  often  before  the  other  organisms 
have  had  time  to  develop  to  any  great  extent.  The  flasks 
which  show  the  presence  of  vibrios  are  used  to  inoculate 
agar  and  gelatine  plates,  a  loopful  of  the  fluid  being 
withdrawn  from  the  surface  for  this  purpose.  Suspicious 
colonies  on  the  agar  and  gelatine  plates  are  subcultivated 
upon  agar  slopes,  and  their  characters  studied  in  pure 
culture. 

Care  must  be  taken  by  employing  all  available  tests 
such  as  Pfeiffer's  test,  haemolysis  test,  &c,  to  determine 
that  the  vibrio  isolated  is  a  true  cholera  organism,  and 
not  one  of  the  closely  allied  vibrios. 

Interpretation  of  Results. — In  the  interpretation  of  the 
results  of  the  bacteriological  examination  of  a  water,  it 
is  usual  to  have  some  sort  of  a  rough  standard  of  purity, 
and  for  that  purpose  the  following  table  for  temperate 
climates  may  be  quoted  ;  but  it  must  be  remembered 
that  in  the  Tropics  the  ordinary  water  organisms  grow 
more  readily  at  the  higher  temperatures  than  in  England, 
so  that  the  counts  on  gelatine  cultivated  at  220  C.  differ 
little  from  those  on  agar  grown  at  37°  C. 


HYPHOMYCETES  417 

(a)  Deep  Waters. 

•     {Springs  and  deep  wells.) 

Gelatine  count        ...         ...     Not  over  50  organisms  per  c.c. 

Agar  count  ...  ...  ...  „         10  ,,  „ 

B.  colt  communis    ...         ...     Should  be  absent  from  100  c.c. 

(b)  Surface  Waters. 

(e.g.,  rivers  for  drinking  purposes,  shallow  wells,  upland 
surface  waters.) 

Gelatine  count        ...         ...     Not  over  500  organisms  per  c.c. 

Agar  count  ...  ...  ...  „  50  ,,  „ 

B.  coli  communis   ...         ...     Should  be  absent  from  10  c.c. 

It  is  hardly  necessary  to  add  that  should  the  examina- 
tion reveal  the  presence  of  either  the  B.  typhosus  or 
Koch's  comma  bacillus,  the  water  should  be  condemned 
forthwith. 

HYPHOMYCETES    (MOULDS). 

Of  the  various  fungi  which  attack  the  skin  and  hair, 
some  are  widely  distributed,  though  more  common  and 
luxuriant  in  their  growth  in  the  Tropics.  Pityriasis 
versicolor  comes  under  this  head.  Others,  like  Favus, 
may  be  common  in  some  places,  but  as  in  temperate 
climates,  are  of  limited  distribution.  The  commoner 
fungi  attacking  the  hair  of  the  head  are  unknown  in 
many  tropical  countries.  The  fungus  attacking  man 
at  present  recognized  as  peculiar  to  the  Tropics  is  a 
cutaneous  ringworm,  Tinea  imbricata,  characterized 
clinically  by  the  large  size  of  the  epidermal  scales,  which 
are  partially  detached,  and  the  tendency  to  form  geo- 
metrical patterns. 

For  the  demonstration  of  the  fungi  causing  these 
various  affections,  the  older  method  consisted  in  soaking 
the  hair  or  scales  in  a  7  per  cent,  solution  of  caustic 
potash,  which  rendered  the  keratin  clear  and  transparent, 
whilst  the  fungus  was  less  affected,  and  could  be  clearly 
seen.  This  method  causes  swelling  of  the  fungus  and 
27 


418  KtNGI 

spores,  and  therefore  is  not  suited  for  the  differentiation 
of  the  varieties  or  species  of  fungi. 

A  modification  of  Gram's  method  of  staining  gives 
more  useful  results,  but  is  slow.  The  hair  or  scale  is 
stained  in  aniline  gentian  violet  for  five  minutes,  and 
dried  with  blotting  paper.  It  is  then  treated  with  Gram's 
iodine  solution  for  two  minutes,  and  again  dried  with 
blotting  paper.  It  is  then  covered  with  aniline  oil,  to 
which  a  little  iodine  has  been  added,  and  left  till  the 
fungus  can  be  seen.  It  should  be  examined  from  time 
to  time  under  the  microscope,  as,  though  the  process 
is  slow,  ultimately  even  the  fungus  will  be  decolourised. 
Do  not  wait  till  all  the  tissue  is  clear,  but  when  nearly 
so,  treat  with  aniline  oil,  and  clear  in  xylol.  Mount 
in  xylol  balsam. 

The  points  to  observe  are  the  arrangement  of  the 
growth,  whether  inside  or  outside  the  hair,  scale,  &c, 
the  presence  and  the  size  of  the  spores.  The  nomencla- 
ture of  these  fungi  is  based  on  these  points.  According 
to  the  seat  of  growth  of  the  fungus  it  is  an  ecto-  or  enclo- 
thrix,  and  microsporon  or  megalosporon  according  to  the 
size  of  the  spores. 

These  ringworms  are  true  fungi.  The  fungi  are  multi- 
cellular organisms  composed  of  filaments,  either  simple 
or  branched,  or  jointed  or  unjointed.  These  filaments 
are  called  hyphce,  and  if  they  project  into  the  air  are  aerial 
hyphce,  or  down  into  the  substance  of  the  medium  they 
are  known  as  sub-aerial  hyphce. 

They  frequently  form  a  compact  mass — a  mycelium — 
and  if  this  form  a  hard,  dense  mass  it  is  known  as  a 
sclerotium.  Sexual  reproduction  as  well  as  reproduc- 
tion by  fission  has  been  proved  to  occur  in  most  members 
of  the  group. 

These  fungi  include  the  ordinary  moulds,  and  some, 
such  as  ergot,  form  compounds  which,  when  eaten,  are 
poisonous. 

In  addition  to  the  cutaneous  fungi  which  cause  the 
true  ringworms  (Tinea),  fungi   may  be  found  in  mouth, 


FUNGI 


419 


ear  or  nose,  as  well  as  in  pulmonary  cavities.  These  are 
secondary  growths.  In  bird-rearers,  who  take  uncooked 
grain  in  their  mouths,  a  true  pneumono-mycosis  occurs, 
and  in  the  Tropics  a  similar  pulmonary  disease  simu- 
lating tuberculosis  occurs. 


Fig.  159  (after  Eyre). — A,  Aspergillus,  a,  mycelium  ;  b,  hypha  ;  c,  slerig- 
mata ;  d,  spore.  B,  Penicillium.  a,  mycelium  ;  b,  hypha;  c,  basidia  ; 
d,  sterigma  ;  e,  spore,  c,  Mucor.  a,  mycelium  ;  b,  hypha;  c,  columella; 
d,   sporagium  ;  e,  spores. 


It  is  possible  that  some  cases  of  madura  foot,  the  black 
variety,  are  due  to  a  fungus  and  not  to  a  streptothrix. 

The  tropical  fungi,  aerial  and  otherwise,  have  so  far 
received  little  attention  and  should  offer  a  fruitful  held 


420  FUNGI 

for  research.  Most  of  the  fungi  grow  readily  on  nutrient 
media,  but  best  if  a  sugar  be  added.  Maltose  is  the  most 
suitable  for  the  fungi  of  the  ringworms. 

Many  of  the  aerial  fungi  will  not  grow  at  high  tem- 
peratures, such  as  blood  heat,  though  they  flourish  at 
lower  temperatures. 

Of  the  commoner  moulds,  mention  may  be  made  of 
Mucor,  Aspergillus  and  Penicillium. 

Mucor  is  the  common  white  mould  frequently  seen  on 
bread,  jam,  &c,  and  is  a  common  contamination  of 
plate  cultures.  It  is  not  known  to  cause  disease  in  man, 
but  is  occasionally  found  as  an  epiphytic  growth  in  the 
externa]  auditory  canal,  bronchiectatic  cavities,  &c. 

Asexual  reproduction  takes  place  by  a  filament  or 
hypha  growing  upwards.  At  its  apex  a  septum  forms 
and  then  a  globular  swelling  appears — sporagium.  This 
possesses  a  definite  membrane.  From  the  septum  grows 
a  club-shaped  mass  of  protoplasm-columella.  The  rest 
of  the  contained  protoplasm  breaks  up  into  spores. 
Finally  the    membrane  ruptures    and  the    spores    escape 

(fig.  159-  C). 

Aspergillus  is  another  common  free-living  form  and  is 
occasionally  associated  with  disease  in  man.  It  may 
occur  in  the  lung,  especially  in  bird-fanciers,  causing 
pneumono-mycosis.  One  form  of  madura  foot  is  be- 
lieved to  be  caused  by  a  variety  of  this  fungus.  Some 
authorities  maintain  that  pellagra  is  due  to  Aspergillus 
fumigatus  contaminating  damaged  maize  used  as  food. 

Asexual  reproduction  is  as  follows: — A  filament  grows 
upwards  and  its  termination  becomes  clubbed  :  on  the 
clubbed  extremity  flask-shaped  cells  appear — sterigmata. 
At  the  free  end  of  each  sterigma  are  formed  oval  bodies — 
the  spores — which  when  ripe  are  thrown  off  from  the 
sterigma  (fig.  159,  a). 

Penicillium  is  a  common  green  mould  found  growing 
on  damp  bread  or  jam  and  is  not  known  to  be  associated 
with  any  human  disease. 

Asexual  reproduction  takes  place  by  a  filament  growing 


YEASTS 


421 


upwards — goniodophore — and  its  apex  dividing  into 
several  branches  called  basidia.  At  the  apex  of  each 
branch  a  flask-shaped  cell  or  sterigma  appears.  At  the 
apex  of  each  sterigma  appears  a  row  of  oval  cells  forming 
the  spores.  These  when  ripe  are  cast  off  from  the 
sterigmata  (fig.  159,  b). 

Blastomycetes  (Yeasts). 

Yeasts,  or  blastomycetes,  are  frequently  found  in  the 
mucous  cavities  and  occasionally  in  ulcers  or  other  skin 
lesions.  They  are  distinguished  from  bacteria  not  only 
by  the  method  of  reproduction  but  also  by  their  greater 
size.  In  some  species  endospores  are  formed,  but  these 
are  multiple  in  each  cell  and  not  single  as  in  bacteria. 


-  B 


Fig.  160  (after  Eyre). — A,  Torula  ;  a,  mother  eell  ;  b,  bud  ;  c,  secondary 
bud.  B,  Saccharomycetes  ;  a,  mother  cell  ;  b,  bud  ;  c,  vacuole  ;  d,  cell-forming 
spores ;  e,  spores. 

The  yeasts  may  be  divided  up  into  two  groups  accord- 
ing as  they  are  able  to  produce  spores  or  not. 

These  two  groups  are  known  as  saccharomycetes  and 
torulae.  Both  reproduce  by  budding,  but  in  the  saccharo- 
mycetes or  true  yeasts  there  is  an  alternative  method  of 
reproduction,  namely,  by  the  formation  of  spores,  each 
cell  giving  rise  to  four  spores  arranged  like  a  pyramid  of 
billiard  balls  (fig.  160,  B).     The  torulae  never  form  spores. 

Old  cultures  of  yeasts  frequently  form  films  in  which 
the  individual  cells  become  much  elongated,  like  those  in 
the  mycelium  of  a  mould. 

The  yeasts  are  of  considerable  interest,  as  alcoholic 
and  other  forms  of  fermentation  are  due  to  their  agency. 


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426  water  analysis— ikon 

The  Chemical  Analysis  of  Water. 

The  methods  employed  for  the  examination  of  a 
water  to  determine  its  suitability  or  otherwise  for  drink- 
ing and  domestic  purposes  are  usually  four  in  number, 
and  it  is  only  by  the  employment  of  all  these  methods 
that  a  true  and  accurate  opinion  can  be  expressed  as  to 
the  potability  and  fitness  for  domestic  use  of  any  water 
under  consideration. 

These  four  methods  are: — 

(i)  Physical. 

(2)  Biological. 

(3)  Bacteriological. 

(4)  Chemical. 

By  a  Physical  examination  we  determine  the  turbidity, 
colour,  odour  and  taste. 

The  Biological  examination  enables  one  to  determine 
the  presence  or  absence  of  many  lowly  organisms,  bac- 
terial and  protozoal,  and  the  ova  and  larvae  of  intestinal 
worms  and  other  parasites. 

The  Bacteriological  examination  is  of  the  utmost  im- 
portance. By  this  means  pathogenic  bacteria,  if  present, 
may  usually  be  detected,  or  bacteria  may  be  found,  the 
presence  of  which  points  conclusively  to  sewage  pollution. 

The  Chemical  examination  of  water  will  frequently  give 
sufficient  data  on  which  to  condemn  a  water  for  pota- 
bility or  domestic  usage. 

In  any  chemical  analysis  of  water  the  principal  chemi- 
cal substances  which  have  to  be  sought  for,  and  if  found, 
quantitatively  estimated,  are  as  follows  : — 

(1)  Poisonous  metals ;  (2)  Free  ammonia  ;  (3)  Albu- 
minoid ammonia  ;  (4)  Nitrates  ;  (5)  Nitrites  ;  (6)  Chlor- 
ides ;  (7)   Hardness. 

Metals  in  the  form  of  salts  are  occasionally  found  in 
water.  Chief  among  these  are  iron  and  lead,  and  more 
rarely  copper,  zinc,  tin,  and  arsenic. 

The  usual  tests  for  these  metals  are  as  follows  : — 

Iron  (Qualitative). — Place  50  c.c.  of  the  water  in  a 
white   porcelain  dish.     Next  dip  a   clean  glass   rod  into 


IKON — COPPER  427 

ammonium  sulphide  and  draw  the  rod  through  the  water 
in  the  dish.  If  iron  is  present  a  dark  colouration  or 
streak  will  be  formed  along  the  track  of  the  rod,  and  will 
be  more  or  less  intense  in  colour,  according  to  the  amount 
of  iron  present  in  the  sample.  A  few  drops  of  hydro- 
chloric acid,  if  added,  will  discharge  this  colouration. 

It  on  (Quantitative). — Place  50  c.c.  of  the  sample  water 
in  a  Nessler  glass  :  next  add  one  or  two  drops  of  ammo- 
nium sulphide.  This  will  produce  a  brownish-black 
colouration.  Into  a  second  Nessler  glass  measure  50  c.c. 
of  distilled  water,  adding  one  or  two  drops  of  ammonium 
sulphide,  and  carefully  run  into  this  mixture  from  a 
burette,  drop  by  drop,  a  standard  solution  of  ferric  chlor- 
ide, of  such  a  strength  that  1  c.c.  equals  'i  mgm.  of  Fe, 
until  the  colour  matches  that  of  the  water  sample.  Note 
the  quantity  of  standard  solution  used,  and  by  a  simple 
calculation  the  number  of  milligrammes  of  Fe  in  the 
50  c.c.  of  sample  water  may  easily  be  determined. 

The  result  should  be  expressed  in  parts  per  100,000. 

The  standard  iron  solution  is  prepared  by  dissolving 
1*0004  grm.  of  iron  wire  in  nitro-hydrochloric  acid, 
precipitate  with  ammonia,  wash  and  re-dissolve  the  ferric 
oxide  in  a  little  pure  HC1  and  dilute  to  ten  litres. 
1  c.c.  of  this  solution  equals  "i  mgm.  of  Fe. 

Iron,  although  hardly  a  poisonous  metal,  if  in  con- 
siderable quantities  in  a  water,  renders  it  unsuitable  for 
drinking  purposes  owing  to  its  nauseous  taste.  Any 
urater  containing  more  than  1  gr.  of  Fe  per  gallon  is 
unfit  for  use. 

Lead  (Qualitative). — This  test  is  performed  in  the  same 
way  as  the  one  described  for  the  detection  of  iron.  The 
brownish-black  colouration  produced  is,  however,  un- 
altered by  the  addition  of  a  few  drops  of  HC1  and  of  KCN. 

Lead  (Quantitative). — The  estimation  of  the  amount  of 
lead  present  is  made  in  the  same  manner  as  that  described 
for  iron,  by  substituting  a  standard  solution  of  lead 
acetate  for  the  standard  solution  of  ferric  chloride  used 
in  the  estimation  of  that  metal. 


428  COPPER — TIN 

Standard  lead  solution  is  made  by  dissolving  with 
the  aid  of  acetic  acid  1*83  grra.  of  lead  acetate  in  ten 
litres  of  distilled  water.  1  c.c.  of  this  solution  equals 
•1  mgm.  of  Pb. 

Copper  (Qualitative). — The  presence  of  this  metal  is 
detected  by  the  same  means  as  have  been  described  for 
the  detection  of  iron  and  lead.  The  colouration  pro- 
duced is  unchanged  on  the  addition  of  HC1,  but  is 
destroyed  on  adding  KCN. 

Copper  (Quantitative). — Place  50  c.c.  of  the  sample 
water  in  a  Nessler  glass  and  add  a  few  drops  of  HC1, 
and  sufficient  of  a  solution  of  potassium  ferrocyanide 
to  produce  the  maximum  colouration.  Into  a  second 
Nessler  glass  measure  50  c.c.  of  distilled  water,  a  few 
drops  of  HC1  and  about  1  c.c.  of  the  potassium  ferro- 
cyanide solution,  and  add  from  a  burette,  drop  by  drop, 
a  sufficient  amount  of  a  standard  solution  of  copper 
sulphate  to  match  the  colour  in  the  first  glass.  By  noting 
the  amount  of  standard  solution  used  to  produce  this 
result,  it  is  easy  to  determine  the  amount  of  copper 
present  in  the  sample. 

Standard  solution  of  copper  sulphate  is  made  by  dis- 
solving 3*95  grm.  of  copper  sulphate  in  ten  litres  of  dis- 
tilled water.     1  c.c.  of  this  solution  equals  "i  mgm.  of  Cu. 

Zinc  (Qualitative). — Place  10  c.c.  of  the  sample  water  in 
a  test  tube  and  add  a  few  drops  of  ammonium  hydrate  to 
render  it  slightly  ammoniacal.  Boil  and  filter.  A  few 
drops  of  potassium  ferrocyanide  added  to  the  filtrate  will 
give  a  white  gelatinous  precipitate  of  zinc  ferrocyanide  if 
zinc  be  present. 

Zinc  (Quantitative). — A  measured  quantity  of  the  water, 
concentrated  if  necessary,  is  treated  with  a  few  drops  of 
ammonium  sulphide,  causing  a  precipitate  of  sulphide 
of  zinc.  This  precipitate  is  collected  by  filtration,  well 
washed  with  dilute  ammonium  sulphide,  dried,  ignited 
at  a  bright  red  heat,  cooled  and  weighed  as  zinc  oxide 
(ZnO  x  o-8  =  Zn). 

Tin  (Qualitative). — Evaporate  one  litre  of  the  water  to 


TIN — ARSENIC  429 

a  small  bulk,  acidulate  with  HC1  and  saturate  with  H2S 
in  a  white  porcelain  dish.  A  yellow  precipitate  of  stannic 
sulphide,  in  the  absence  of  other  heavy  metals,  would 
indicate  the  presence  of  tin. 

As  confirmatory  evidence  the  brucine  test  as  here 
described  may  be  used  : — 

A  solution  of  brucine  prepared  as  follows  is  required  : 
•5  grm.  of  brucine  are  dissolved  in  5  c.c.  of  pure 
HNO3  in  the  cold,  250  c.c.  of  water  is  added  and  the 
whole  boiled  for  fifteen  minutes.  Sufficient  water  is 
afterwards  added  to  make  the  total  bulk  up  to  250  c.c. 
100  c.c.  of  the  water  to  be  tested  is  taken  and  evaporated 
to  dryness.  The  residue  is  dissolved  in  a  few  drops  of 
distilled  water  and  1  c.c.  of  the  brucine  solution  is  added. 
If  tin  be  present,  a  reddish-violet  colour  will  be  produced. 

Tin  (Quantitative). — One  litre  of  the  water  is  taken  and 
slowly  evaporated  on  a  water  bath  to  a  small  bulk  (say 
50  c.c). 

This  is  then  acidulated  with  HC1  and  finally  saturated 
with  H2S.  Tin,  if  present,  will  then  be  precipitated  as 
yellow  stannic  sulphide. 

This  precipitate  is  collected  and  treated  with  strong 
nitric  acid,  forming  meta-stannic  acid.  This  product  is 
then  ignited,  producing  stannic  oxide. 

By  weighing  this  oxide  the  amount  of  tin  present  in 
the  water  used  can  be  estimated  (Sn02  X  0*785  =  Sn). 

Tin  is  more  frequently  found  in  meat  essences  and 
food  contained  in  tins.  The  contents  must  be  evapo- 
rated to  dryness  and  ignited.  An  excess  of  HC1  should 
then  be  added  and  again  evaporated  to  dryness.  The 
residue  is  dissolved  in  water  acidified  with  HC1  and 
saturated  with  H2S. 

Arsenic. — To  detect  arsenic  in  water  a  litre  of  the  water 
is  rendered  alkaline  by  solid  sodium  carbonate,  evapo- 
rated nearly  to  dryness  and  then  introduced  into  Marsh's 
apparatus. 

Any  water  in  which  any  of  these  metals,  except  iron, 
are  detected,  should  be  unhesitatingly  condemned. 


430  FREE    AMMONIA 

The  determination  of  free  ammonia,  albuminoid  am- 
monia, nitrates,  nitrites  and  chlorides,  is  of  importance, 
as  these  substances,  if  in  considerable  amount,  usually 
indicate  contamination  with  organic  matter,  possibly 
sewage.  In  moderate  quantities  they  are  not  in  them- 
selves injurious. 

Free  Ammonia. — The  method  used  for  the  estimation 
of  this  substance  is  a  colori metric  one,  and  is  known  as 
Wanklyn's  process. 

Into  a  glass  distilling  flask,  of  about  a  litre  capacity, 
connected  with  a  condenser,  pour  500  c.c.  of  the  water 
and  add  a  pinch  of  sodium  carbonate  and  a  small  piece  of 
pumice-stone.  The  flask  is  then  heated  over  a  Bunsen 
flame,  and  three  Nessler  glasses,  of  50  c.c.  each  of  the 
distillate,  are  collected.  This  amount  of  distillate,  viz., 
150  c.c,  is  found  to  be  sufficient  to  obtain  all  the  free 
ammonia  in  any  sample  of  water. 

Into  the  first  of  these  distillates  2  c.c.  of  Nessler's  solu- 
tion are  introduced  from  a  burette,  when  a  yellow  colour 
will  be  produced  of  a  greater  or  lesser  intensity,  accord- 
ing to  the  amount  of  free  ammonia  present.  This 
colouration  is  then  matched  in  another  Nessler  glass, 
using  distilled  water,  2  c.c.  of  Nessler's  solution,  and  to 
this  a  sufficient  measured  quantity  of  a  standard  solu- 
tion of  ammonium  chloride  is  added  till  the  colouration 
matches.  This  standard  solution  is  made  by  dissolving 
3"  14  grm.  of  anhydrous  ammonium  chloride  in  a  litre  of 
distilled  water,  again  diluted  one  hundred  times,  so  that 
1  c.c.  of  the  resultant  fluid  =  -oi  mgm.  of  ammonia. 

The  second  and  third  distillates  are  treated  in  the 
same  way,  and  the  total  amount  of  standard  solution  of 
NH^Cl  used,  is  noted. 

As  each  cc.  of  standard  solution  equals  -oi  mgm.  of 
NH3,  it  is  a  simple  matter  to  calculate  the  amount  of  free 
ammonia  present  in  the  original  500  c.c.  of  water  taken, 
and  from  that  to  express  the  result  in  parts  per  100,000. 

Nessler's  solution  is  prepared  as  follows  : — 

To    800    c.c.  of    distilled    water    are    added   35    grm. 


ALBUMINOID   AMMONIA — NITRITES  431 

of  potassium  iodide  and  13  grm.  of  perchloride  of 
mercury.  This  is  then  boiled  and  well  stirred  until  the 
salts  are  dissolved.  Cold,  saturated  solution  of  HgCl2 
is  next  added,  until  a  permanent  red  precipitate  appears. 
120  grm.  of  sodium  hydrate  are  now  added  and  the 
whole  made  up  to  1  litre  with  water.  The  solution  is 
then  rendered  sensitive  by  the  addition  of  a  little  more 
of  the  solution  of  HgCl2. 

Albuminoid  Ammonia. — To  the  residue  left  in  the  dis- 
tilling flask  after  the  last  process  add  50  c.c.  of  alkaline 
permanganate  solution  (K2Mn04  8  grm.,  NaOH  200 
grm.,  to  a  litre  of  distilled  water),  and  proceed  to 
distil  over  as  before,  continuing  the  operation  until  no 
more  ammonia  comes  over. 

The  determination  of  the  amount  of  ammonia  in  this 
case  is  conducted  in  precisely  the  same  manner  as  for 
the  free  ammonia. 

Nitrites  (Qualitative). — Make,  with  distilled  water,  a  5 
per  cent,  solution  of  meta-phenylene  diamine. 

Decolourise  by  shaking  up  with  animal  charcoal  and 
filter.  The  solution  should  now  be  colourless.  If  this 
is  not  so,  repeat  the  treatment  with  animal  charcoal  until 
such  result  is  obtained. 

To  100  c.c.  of  the  sample  water  in  a  Nessler  glass  add 
a  few  drops  of  dilute  sulphuric  acid  and  then  about  1  c.c. 
of  the  meta-phenylene  diamine  solution,  and  place  the 
glass  in  a  warm  place  for  half  an  hour.  By  the  end  of 
that  time  a  yellow  colour  will  be  produced  if  the  water 
contains  nitrites. 

Nitrites  (Quantitative). — A  standard  solution  of  potas- 
sium nitrite  is  used  of  such  a  strength  that  1  c.c.  equals 
'oi  mgm.  N.  This  is  made  by  dissolving  ri  grm.  of 
pure  silver  nitrite  in  boiling  distilled  water.  To  this  is 
added  KC1,  which  precipitates  the  silver  as  AgCl.  The 
whole  is  then  made  up  to  one  litre  and  the  silver  allowed 
to  settle.  From  the  clear  supernatant  liquid  100  c.c.  are 
taken  and  made  up  to  a  litre  with  distilled  water.  This 
solution  will  be  of  the  required  strength. 


432  NITRATES 

By  using  this  standard  solution  the  colour  produced  in 
the  ioo  c.c.  of  sample  water  with  the  meta-phenylene 
diamine  may  be  exactly  matched  in  the  same  quantity 
of  distilled  water.  Knowing  the  amount  of  standard 
solution  required  to  produce  this  result,  a  simple  calcula- 
tion is  then  all  that  is  needed  to  determine  the  amount  of 
nitrites  in  the  water  subjected  to  analysis. 

Nitrates  (Qualitative). — Take  10  c.c.  of  the  sample  water 
in  a  test  tube  and  to  this  add  about  I  c.c.  of  a  saturated 
solution  of  brucine  and  well  mix  by  shaking.  Now 
carefully  introduce  down  the  side  of  the  tube  with 
a  pipette  about  2  c.c.  of  pure  sulphuric  acid,  so  that  the 
acid  forms  a  distinct  layer  beneath  the  mixture  of  water 
and  brucine.  If  nitrates  are  present  in  the  water  under 
examination,  a  pink  ring  changing  to  one  of  a  brownish- 
yellow  colour  will  be  seen  at  the  junction  of  the  two 
liquids. 

Nitrates  (Quantitative). — The  most  convenient  way  of 
estimating  the  amount  of  nitrates  in  a  water  is  by  the 
process  known  as  the  phenol  sulphonic  acid  method. 

P^or  this  the  following  solutions  are  required  : — 

(1)  Phenol  Sulphonic  Acid. — This  is  made  by  mixing  12 
grm.  of  pure  phenol  with  6  c.c.  of  distilled  water  and 
74  c.c.  of  pure  sulphuric  acid,  and  digesting  the  mixture 
for  two  hours  at  ioo°  C. 

(2)  Standard  solution  of  Potassium  Nitrate. — Made  by 
dissolving  722  grm.  of  dried  potassium  nitrate  in  one 
litre  of  distilled  water.     1  c.c.  of  this  equals  -i  mgm.  N. 

The  process  is  thus  carried  out  :  10  c.c.  of  the  sample 
water  and  10  c.c.  of  the  standard  solution  of  potassium 
nitrate  are  evaporated  separately  to  dryness  in  two 
porcelain  dishes  over  a  water  bath. 

To  each  of  the  residues  1  c.c.  of  the  phenol  sulphonic 
acid  is  added,  and  the  dishes  allowed  to  remain  on  the 
bath  for  a  few  minutes. 

The  contents  of  the  dishes  are  now  washed  out  succes- 
sively with  about  20  c.c.  of  distilled  water  into  two  Nessler 
glasses,  and  20  c.c.  of  liquor  ammonias  added  to  each,  the 


CHLOK'IDES  433 

whole  amount  of  liquid  in  each  glass  being  made  up  to 
ioo  c.c.  with  distilled  water. 

Any  nitrates  in  the  solutions  act  on  the  phenol  sul- 
phonic  acid,  converting  it  into  picric  acid,  which  is  again 
turned  by  the  ammonia  into  ammonium  picrate.  This 
gives  a  yellow  colour  to  the  solution,  the  intensity  of  the 
same  being  proportional  to  the  amount  of  nitrate  present. 
Now  take  a  third  Nessler  glass  and  pipette  into  it  a 
sufficient  quantity  of  the  darker  solution,  which  when 
diluted  with  distilled  water  up  to  ioo  c.c.  will  exactly 
match  in  colour  the  more  lightly  coloured  solution. 
Assuming  that  5  c.c.  of  the  contents  of  the  Nessler  glass 
containing  the  standard  solution  when  diluted  up  to 
100  c.c.  exactly  match  the  water  sample,  then  the  latter 
must  contain  T^y  of  10  c.c.  of  standard  solution. 

That  is  to  say,  10  c.c.  of  the  sample  water  contains  an 
amount  of  nitrates  equivalent  to  that  contained  in  "5  c.c. 
of  the  standard  solution.  The  exact  strength  of  the  latter 
being  known,  it  is  easy  to  work  out  the  parts  per  100,000 
of  nitrates  in  the  water  under  consideration. 

Chlorides  (Qualitative). — To  50  c.c.  of  the  water  in  a 
Nessler  glass  add  a  few  drops  of  nitric  acid  and  then  a 
little  silver  nitrate  solution.  If  chlorides  are  present  this 
gives  a  white  haze,  or  a  precipitate  if  they  are  abundant. 

Chlorides  (Quantitative). — Two  reagents  are  used  in  the 
estimation  of  chlorides. 

(1)  A  5  per  cent,  solution  of  potassium  chromate. 

(2)  A  standard  solution  of  silver  nitrate,  made  by  dis- 
solving 4*8  grm.  of  AgN03  in  a  litre  of  distilled  water. 
1  c.c.  of  this  solution  =  1  nigra.  CI. 

Place  103  c.c.  of  the  water  under  examination  in  a 
white  porcelain  dish,  and  add  1  c.c.  of  the  KCr04  solu- 
tion and  stir.  Whilst  constantly  stirring  with  a  glass 
rod,  run  in  from  a  burette,  drop  by  drop,  the  silver 
nitrate  solution,  until  the  yellow  colour  becomes  perma- 
nently orange.  Now7  read  off  on  the  burette  the  amount 
of  silver  nitrate  solution  used,  and  calculate  from  this 
the  amount  of  chlorides  present  in  the  sample  water. 
28 


434  CHLORIDES 

Hardness  in  the  water  is  either  temporary  or  perma- 
nent. The  temporary  hardness,  which  may  be  got  rid 
of  by  boiling,  is  produced  by  calcium  carbonate  and 
magnesium  carbonate,  held  in  solution  by  the  action  of 
C02.  The  permanent  hardness  consists  principally  of 
some  sulphates,  chlorides  and  nitrates  of  calcium  and 
magnesium. 

The  total  hardness  is  the  sum-total  of  both  the  tem- 
porary and  permanent  hardness. 

The  usual  way  of  estimating  the  amount  of  total  hard- 
ness is  by  the  application  of  what  is  known  as  Clark's 
process. 

A  standard  soap  solution,  made  with  equal  parts  of 
spirit  and  water,  is  used,  of  such  a  strength  that  i  c.c 
exactly  neutralizes  i  mgm.  of  calcium  carbonate. 

Take  ioo  c.c.  of  the  sample  water  in  a  200  c.c.  stoppered 
bottle,  and  run  in  the  soap  solution  1  c.c.  at  a  time, 
shaking  well  after  each  addition,  until  a  lather  ^  in. 
thick  remains  unbroken  for  five  minutes.  Read  off  the 
number  of  cubic  centimetres  of  soap  solution  used  and 
deduct  1  c.c,  as  being  necessary  for  the  production  of  a 
lather  in  100  c.c.  of  distilled  water.  The  remainder  will 
then  give  the  amount  of  hardness  present,  expressed  in 
parts  per  100,000. 

In  expressing  an  opinion  as  to  the  suitability  or  other- 
wise of  any  water  for  drinking  or  domestic  purposes,  a 
careful  survey  of  all  the  factors  revealed  by  the  chemical 
analysis  must  be  made,  combined  with  a  critical  exami- 
nation of  the  source  of  the  supply  and  of  all  vessels  used 
in  the  storage  of  the  same. 

It  is  impossible  to  lay  down  any  hard  and  fast  standard 
of  purity  for  a  water,  and  every  case  must  be  judged  on 
its  merits. 

Briefly,  it  may  be  stated  that  any  water  containing  any 
nitrites,  indicating  recent  pollution,  should  be  condemned 
forthwith.  The  same  applies  to  any  water  containing 
any  poisonous  metal. 

The  amount  of  hardness  in  water  varies  between  wide 


HARDNESS  435 

limits.  A  figure  representing  thirty  or  more  degrees 
of  total  hardness  would  condemn  a  water  as  unfit 
for  drinking  or  domestic  purposes,  unless  that  figure 
could  be  considerably  reduced  by  a  suitable  process  of 
softening. 

The  amount  of  both  free  and  albuminoid  ammonia, 
unless  in  exceptional  abundance,  does  not  necessarily 
condemn  a  water.  Some  waters,  e.g.,  rain  water,  may 
give  a  high  figure  for  free  ammonia,  but  the  albuminoid 
ammonia  will  be  small.  On  the  other  hand,  peaty  water 
may  give  a  figure  as  high  as  'oi  per  100,000  for  the 
albuminoid  ammonia,  but  the  free  ammonia  present 
will,  in  the  absence  of  pollution,  be  practically  a  negli- 
gible quantity. 

In  all  cases  where  the  figures  for  either  or  both  of  the 
ammonias  are  high,  these  must  be  considered,  in  con- 
junction with  the  figures  of  the  nitrates  and  chlorides, 
before  giving  an  opinion.  A  water  containing,  say,  '005 
parts  per  100,000  or  more  free  ammonia,  and  '01  parts 
per  100,000  or  more  of  albuminoid  ammonia,  should 
excite  grave  suspicion.  If,  at  the  same  time,  the  figures 
obtained  for  chlorides  and  nitrates  were  also  high,  e.g., 
CI  5  parts  per  100,000,  and  nitrates  "4  parts  per  100,000, 
such  water  should  be  unhesitatingly  condemned. 

A  high  chloride  figure  must  always  be  carefully  investi- 
gated as  it  may  indicate  contamination  with  sewage  or 
urine.  Many  waters  contain  considerable  quantities  of 
chlorides,  as  in  waters  derived  from  the  lower  green- 
sands,  and  deep  down  in  the  chalk,  which  are  harmless. 
An  exceptionally  high  figure  for  chlorides  in  a  well  near 
the  sea  indicates  that  the  same  is  polluted  by  the  sea 
water,  and  the  water  therefrom  is  unpalatable. 

REFERENCES. 

Notter  :  "  Theory  and  Practice  of  Hygiene." 
Somerville  :  "  Practical  Sanitary  Science." 
Whitelegge:  "  Hygiene  and  Public  Health." 
Fresenius  :  "  Quantitative  Analysis." 
Sutton  :  "  Volumetric  Analysis." 


436 


CHAPTER  XXIII. 

Measurements. 

MEASUREMENTS  of  the  various  eggs,  parasites  and 
normal  and  abnormal  cells,  are  of  considerable  import- 
ance and  are  easily  made. 

The  simplest  and  most  satisfactory  method  of  micro- 
scopic measurement  is  by  drawing  to  scale,  which  can 
be  readily  done  by  the  use  of  a  camera  lucida  or  draw- 
ing camera.  A  micromillimetre  scale  is  used  as  an  object, 
and  with  the  microscope  vertical  or  inclined  at  an  appro- 
priate angle,  depending  on  the  form  of  camera  used,  the 
scale  as  it  appears  through  the  camera  lucida  is  drawn  on 
a  piece  of  paper.  Gower's  haemocytometer  slide,  which 
is  divided  into  T\j  mm.,  or  a  Thoma-Zeiss,  which  is  divided 
into  2Xo  mm.,  may  be  used  instead  of  the  micromillimetre 
scale,  or  any  other  will  suffice.  This  drawing  of  -^  mm. 
must  be  further  subdivided  by  compasses.  This  gives  the 
scale,  and  it  must  be  determined  for  each  objective. 
Provided  that  the  distance  of  the  paper  from  the  camera 
lucida  is  constant,  which  is  best  ensured  by  working 
with  the  microscope- vertical  and  the  paper  on  the  table, 
a  scale  once  drawn  can  always  be  used. 

The  draw  tube  of  the  microscope  must  always  be  the 
same  length. 

To  measure  an  object  all  that  is  needed  is  an  outline 
drawing  through  the  same  camera  lucida,  and  the  appli- 
cation of  the  scale  to  this  drawing  will  give  the  measure- 
ments. 

Another  simple  method  is  by  the  use  of  a  micrometer 
eye-piece,    which    consists    of   a   glass   disc    on   which   a 


MEASUREMENTS  437 

scale  is  drawn,  and  this  placed  in  the  eye-piece  so  as 
to  be  accurately  in  focus  by  the  anterior  lens.  The  disc 
rests  on  the  diaphragm,  which  can  be  moved  so  that  the 
scale  is  sharply  focussed.  A  measured  scale  is  then 
placed  on  the  stage.  As  before,  Gowers'  or  a  Thoma- 
Zeiss  haemocytometer  scale  may  be  used  instead  of  the 
micromillimetre  scale,  and  the  number  of  the  divisions 
in  the  micrometer  eye-piece,  which  corresponds  to  ^  or 
jfo  millimetre,  or  a  multiple  of  these,  with  the  different 
objectives  is  noted.  With  the  tube  at  constant  length  the 
value  of  the  divisions  in  the  micrometer  eye-piece  so  deter- 
mined is  constant  for  each  objective.  In  measuring,  the 
object  to  be  examined  is  placed  under  the  microscope  and 
the  measurements  in  terms  of  the  micrometer  scale  deter- 
mined, and  from  these  the  real  measurements  calculated. 

For  simple  diameters,  the  micrometer  eye-piece  is 
perhaps  the  most  convenient,  but  for  irregularly-shaped 
bodies,  and  particularly  for  such  objects  as  filaria,  the 
use  of  the  camera  lucida  is  easier,  quicker  and  more 
accurate. 

By  either  of  these  methods  all  that  is  required  to 
measure  an  object,  once  the  scales  are  made,  or  the 
equivalent  in  micromillimetres  of  the  eye-piece  scale 
determined,  is  to  change  the  ordinary  eye-piece  either 
for  the  camera  lucida  or  for  the  eye-piece  containing  the 
micrometer  scale. 

Measurements  may  be  represented  as  decimals  or 
fractions  of  a  millimetre,  but  in  many  ways  it  is  more 
convenient  to  take  as  the  standard  x^oo  °f  a  millimetre 
— a  micromillimetre — usually  indicated  by  the  Greek  a*. 

If  no  scale  be  available  to  standardize  the  micrometer 
eye-piece  or  drawings,  results  by  relative  measurements 
can  be  taken  and  subsequently  standardized.  A  con- 
venient rough  standard  is  the  average  diameter  of  a  red 
corpuscle,  which  is  about  7  to  8  //.. 

Estimation  of  the  Number  of  Corpuscles. —  For  the  deter- 
mination of  the  number  of  elements  in  a  given  volume  of 
fluid,   as  for  instance   the   number  of  red  or  white  cor- 


438  BLOOD   COUNTS 

puscles  in  blood,  it  is  usually  necessary  to  dilute  such  a 
fluid  to  a  known  extent  so  that  the  number  of  elements 
in  any  given  volume  can  be  counted. 

Such  a  dilution  may  be  made  in  a  graduated  pipette 
by  drawing  up  a  given  volume  of  fluid  and  as  many  more 
volumes  of  a  diluting  fluid  as  is  necessary,  and  mixing 
well.  Such  a  mixture  can  also  be  conveniently  made 
in  Wright's  tubes,  as  the  absolute  volume  is  immaterial  ; 
all  that  is  required  is  any  volume  and  definite  multi- 
plications of  that  volume  in  order  to  get  the  degree  of 
dilution. 

Common  instruments  used  for  the  purpose  are  the 
pipettes  of  the  Thoma-Zeiss  haemocytometer  (fig.  i6i)- 
These  are  so  graduated  as  to  give  a  dilution  of  i  in  10 
or  i  in  ioo,  but  can  be  used  to  give  dilutions  at 
intervals  of  10  from  i  in  10  to  i  in  ioo,  and  in  intervals 
of  ioo  from  i  in  ioo  to  i  in  1,000  ;  as  the  fluid  last  drawn 
up  into  the  tube  when  the  mark  ior  or  n  is  reached  is  not 
mixed  with  the  blood  but  simply  blown  out  again,  it  does 
not  count  in  the  dilution. 

These  tubes  are  convenient,  and  the  glass  bead  in  the 
mixing  chamber  facilitates  mixing  and  prevents  the 
aggregation  of  corpuscles  into  masses.  The  diluting 
fluid,  when  working  with  blood,  must  be  carefully 
selected  according  to  the  object  to  be  attained.  If  red 
corpuscles  are  to  be  counted  the  fluid  must  be  isotonic 
or  hypertonic,  so  as  to  prevent  the  red  corpuscles  being 
broken  up.  Such  fluids  as  10  per  cent,  solution  of 
sodium  sulphate  are  suitable. 

In  many  cases  it  is  convenient  to  count  the  white 
corpuscles  at  the  same  time,  and  in  that  case  stains  are 
mixed  with  the  diluting  fluid,  which  stain  the  leucocytes 
and  enable  them  to  be  readily  distinguished.  Toisson's 
fluid,  viz.,  glycerine  30  ex.,  sodium  sulphate  8  grm., 
sodium  chloride  1  grm.  methyl  violet  "025  grm.,  and 
water  160  c.c,  is  very  convenient  for  this  purpose, 
but  must  be  filtered  each  time  before  use.  In  other 
cases  where  it  is  not  desired  to  count  the  red  corpuscles 


BLOOD   COUNTS 


439 


and  where  these  may  render  the  enumeration  of  other 
elements  more  difficult,  it  is  better  to  destroy  them.  To 
ensure  their  destruction  it  is  advisable  to  use  a  more 
powerfully  destructive  agent  than  distilled  water,  and 
weak  acetic  acid  is  the  one  generally  employed.  A  i  per 
cent,  solution  of  acetic  acid  is  suitable,  and  to  this  a  little 
methyl  violet  may  be  added  so  as  to  stain  the  leucocytes 
faintly,  thus  rendering  them  more  easily  observable. 

Leucocytes,  &c,  can  be  readily  counted  in  blood  only 
slightly  diluted  when  treated  in  this  manner. 

However  the  dilution  is  made,  the  next  essential  is  to 
obtain  a  definite  measured  volume   of  the  diluted  fluid. 


Fig.  161. — Thoma's  H^mocytometer,  by  Zeiss. 

This  is  done  by  having  a  cell  which,  when  covered  with 
a  cover-glass,  has  a  definite  known  depth.  It  is  also 
further  necessary  to  be  able  to  estimate  the  area  of  the 
base  of  this  cell  or  of  the  portion  of  it  examined.  In 
Gower's  and  in  Thoma-Zeiss'  haemocytometer  (fig.  161) 
this  area  is  determined  by  having  the  side  ruled  in 
squares  with  sides  -^  and  -^  of  a  millimetre  respectively, 
so  that  the  area  is  obtained  by  multiplying  the  sides  of 
the  squares  by  each  other,  and  this  is  multiplied  by  the 
known  depth  of  the  cell,  i.e.,  the  space  between  the 
cover-glass  and  slide,  gives  the  volume  of  the  fluid 
examined. 


44°  I5LOOD    COUNTS 

Instead  of  these  squares  others  use  a  micrometer  eye- 
piece ruled  in  squares.  The  size  of  these  squares  is 
determined  by  comparison  with  a  scale  under  the  micro- 
scope once  for  all. 

In  examining  fluid  for  elements  that  are  scanty  it  is 
often  a  saving  of  time  to  take  the  whole  field  as  the  area 
to  be  examined. 

This  area  is  most  conveniently  determined  by  obtain- 
ing the  diameter  of  the  field  by  observing  the  number 
of  divisions  of  a  scale — a  haemocytometer  scale  again 
will  do — that  form  the  diameter.  To  avoid  fractions  of 
a  division  the  tube  should  be  drawn  out  till  the  diameter 
is  exactly  a  certain  number  of  divisions,  and,  as  pointed 
out  by  Griinbaum,  much  calculation  can  be  avoided  by 
drawing  the  tube  out  till  the  number  of  divisions  is  a 
multiple  of  10. 

The  formula,  it  r2,  then  gives  the  area  of  the  circular 
field.  The  depth  of  the  cell  is  known,  so  that  this  area 
multiplied  by  the  known  depth  gives  the  volume  of 
fluid  examined  in  one  field,  and  this  multiplied  by  the 
number  of  fields  examined  gives  the  total  volume  of 
diluted  fluid  examined.  The  volume  of  original  fluid 
examined  is  obtained  by  dividing  by  the  number  express- 
ing the  degree  of  dilution. 

Dilution  is  merely  for  convenience  and  to  render 
counting  practicable.  In  an  undiluted  fluid,  such  as 
blood,  the  number  of  corpuscles  in  a  small  area,  say 
T^o  mm.  square,  would  be  some  hundreds,  and  therefore 
difficult  to  count,  but  by  diluting  ioo  times  the  number 
will  be  reduced  in  that  area  to  a  dozen  or  less,  a  con- 
venient number  for  counting. 

In  these  calculations  it  is  well  to  avoid  the  exclusive 
use  of  formulae.  A  formula  is  easily  forgotten,  or  only 
in  part  remembered,  and  confusion  and  error  result. 
If  the  calculations  are  made  on  general  principles  there 
is  a  little  waste  of  time,  but  the  possibility  of  error  is 
avoided.  The  dilution  is  made  to  a  known  extent — ten 
times,   a    hundred   times,  or   so  on,  as    is  judged  to  be 


BLOOD   COUNTS  44I 

convenient.  A  known  volume  of  the  diluted  fluid  is 
examined,  represented  by  either  the  area  of  the  field 
multiplied  by  the  depth  of  the  cell,  or  the  area  of  the 
marked  squares  on  the  slide  multiplied  by  the  depth  of 
the  cell  or  the  area  of  the  square  as  seen  in  the  micro- 
meter eye-piece  (previously  determined)  multiplied  by  the 
depth  of  the  cell. 

The  number  of  elements  which  it  is  wished  to  have 
counted  is  determined  in  a  certain  number  of  these 
volumes  of  fluids,  and  the  average  is  taken  by  dividing 
the  total  number  by  the  number  of  volumes  examined. 
The  larger  the  number  of  volumes  taken  the  smaller  is 
the  probable  error. 

All  the  factors  necessary  for  the  calculation  are  thus 
determined,  and  all  that  is  necessary  is  to  reduce  them 
to  comparable  terms,  so  that  the  results  obtained  can  be 
compared  with  other  results.  The  number  of  elements 
is  usually  recorded  as  so  many  per  cubic  millimetre  of 
undiluted  fluid. 

For  example,  suppose  the  blood  has  been  diluted  two 
hundred  times,  and  in  the  area  of  400  squares  of  a  Thoma- 
Zeiss  haemocytometer  there  are  counted  2,500  red  blood 
corpuscles,  or  an  average  of  ^J^q-  =  6*25  per  square. 

Now  each  square  is  -^  mm.,  x  -£>  mm.,  and  the  depth 
of  the  cell  is  ^  mm.,  therefore  one  square  represents  -^ 
x  ^q  x  Txo  c.mm.  of  the  diluted  fluid. 

If  in  3o1oo  c.mm.  of  the  diluted  fluid  there  are  6*25 
corpuscles,  then  in  1  c.mm.  of  the  diluted  fluid  there  are 
6*25  x  4,000  corpuscles.  And  as  the  blood  has  been 
diluted  two  hundred  times,  in  1  c.mm.  of  the  blood  there 
are  6*25  x  4,000  x  200  =  5,000,000  red  blood  corpuscles. 

The  number  of  blood  corpuscles  in  a  cubic  millimetre 
of  blood  may  be  calculated  from  the  formula 

x  =  M 


<i 

in  which  M.  =  the  number  of  corpuscles  counted,  N.  = 
the  dilution  of  the  blood,  and  q  =  the  number  of  small 
squares  counted. 


44-  BLOOD   COUNTS 

This  assumes  that  the  elements  are  uniformly  diffused 
through  the  fluid,  and  every  effort  must  be  made  to 
ensure  this  being  the  ease.  For  blood  there  must  be  no 
delay,  and  the  blood  must  be  mixed  with  the  diluent  as 
quickly  and  thoroughly  as  possible.  Many  of  the  ele- 
ments in  the  blood  and  other  animal  fluids  are  adhesive 
and  tend  to  adhere  in  clumps.  If  these  clumps  are 
present  any  result  obtained  will  be  unreliable. 

Counts  of  red  blood  corpuscles  are  necessary  in  cases 
of  anaemia.  In  some  forms  of  anaemia  the  number  of 
corpuscles  is  not  reduced  and  may  even  be  above  normal. 
In  these  cases  the  haemoglobin  is  much  reduced,  so  that 
on  the  average  each  corpuscle  is  poor  in  haemoglobin. 
Chlorosis  is  the  type  of  this  form  of  anaemia. 

In  others  and  the  more  common  forms  of  anaemia,  in  the 
tropics,  the  number  of  red  corpuscles  is  greatly  reduced, 
sometimes  to  ^  of  the  normal  5,000,000,  and  commonly 
to  between  1,000,000  and  2,000,000.  In  these  forms  of 
anaemia  the  haemoglobin  is  reduced  only  to  about  the 
same  extent  as  the  number  of  the  corpuscles,  so  that, 
on  the  average,  each  red  corpuscle  contains  about  the 
normal  amount  of  haemoglobin,  so  that  they  are  more 
allied  to  pernicious  amvuiia,  though  in  this  form  the 
average  amount  of  haemoglobin  per  red  corpuscle  is 
greater  than  normal. 

In  the  anaemia  resulting  from  haemoglobinuric  fever 
the  number  of  the  red  corpuscles  is  very  rapidly  reduced, 
so  that  in  the  course  of  three  davs  the  red  corpuscles 
will  fall  from  5,000,000  to  1,000,000,  or  even  less,  per 
c.mm.  of  blood.  In  malaria  it  is  unusual  to  find  any 
great  diminution  in  the  number  of  red  corpuscles  as  I  he- 
result  of  an  acute  attack  of  malaria,  though  repeated 
attacks  may  cause  a  considerable  degree  of  anaemia  of 
this  type,  and  even  in  slight  attacks  the  red  corpuscles 
show  more  variation  than  is  normal  in  si/.e,  colour,  and 
shape.  Ross  has  shown  that  even  in  single  paroxysms 
there  is  an  increase  in  urobilin  in  the  faeces,  indicating 
blood  destruction. 


BLOOD   COUNTS  443 

In  anchylostomiasis  in  most  cases  there  is  a  great 
reduction  in  the  number  of  red  corpuscles,  so  great  in 
chronic  progressive  cases  that  only  500,000  red  cor- 
puscles may  be  found  in  a  cubic  millimetre.  There  is 
often,  however,  an  even  greater  fall  in  the  haemoglobin  ; 
so  that  though  in  the  main  this  form  of  anaemia  is  of 
the  type  of  pernicious  anaemia,  in  some  cases  it  is  of 
a  mixed  nature. 

The  forms  of  anaemia  of  the  pernicious  anaemia  type 
are  usually  due  to  blood  destruction  or  hcemolysis.  In 
such  forms  of  anaemia  yellow  pigment  and  iron  deposits 
are  found  in  the  cells  of  the  liver,  kidneys,  &c.  In  many 
cases  of  ankylostomiasis  these  deposits  are  as  extensive 
as  in  cases  of  pernicious  anaemia. 

Various  chemical  agents  can  cause  haemolysis,  and  it 
occurs  as  a  result  of  the  action  of  various  organisms  and 
animal  parasites. 

The  total  leucocyte  counts  should  be  supplemented  by  a 
differential  count  of  the  leucocytes  in  a  well-stained 
film  (vide  p.  58).  By  the  combination  of  these  methods 
the  number  of  leucocytes  of  each  variety  in  a  cubic  milli- 
metre of  blood  can  be  determined. 

Increase  in  the  total  number  of  the  leucocytes  occurs 
in  many  diseases  ;  it  is  known  as  leucocytosis.  Diminu- 
tion in  the  number  of  leucocytes — leucopenia — is  of  less 
importance  unless  marked,  but  occurs  in  some  stages  of 
malaria,  in  kala-azar  and  other  diseases.  Leucocytosis 
is  marked  in  most  cases  of  pneumonia,  and  the  amount 
of  leucocytosis  increases  with  the  severity  of  the  case  to  a 
certain  extent.  In  the  most  severe  attacks,  however,  there 
may  be  no  leucocytosis,  and  therefore  the  absence  of  this 
condition  is  of  even  more  unfavourable  prognosis  than  a 
most  marked  manifestation  of  it. 

In  septic  conditions,  appendicitis  with  suppuration, 
hepatic  abscess,  septic  endocarditis,  &c.,  there  is  well- 
marked  leucocytosis,  though  not  to  the  same  extent  as 
in  some  cases  of  pneumonia.  An  increase  also  occurs 
in    scurvy. 


444  BLOOD  COUNTS 

Even  in  health  there  is  a  considerable  variation — 7,000 
to  10,000 — in  the  number  of  leucocytes,  and  daily  a  varia- 
tion occurs  owing  to  the  increase  in  the  lymphocytes 
during  active  digestion. 

It  is  not  usual  for  the  number  of  the  different  forms 
of  leucocytes  to  be  increased  uniformly.  Usually  some 
forms  arc  increased  and  others  either  increased  to  a  much 
smaller  extent  or  even   diminished. 

In  pneumonia  the  increase  is  mainly  that  of  the  poly- 
morphonuclear leucocytes,  whilst  the  eosinophile  leuco- 
cytes are  not  only  in  smaller  proportion  but  in  smaller 
numbers  than  in  normal  blood.  After  an  attack  of 
malaria  the  leucocytes  are  usually  in  normal  number,  as 
the  larger  mononuclear  leucocytes  arc  increased  and  the 
polymorphonuclear  leucocytes  diminished. 

If  in  addition  to  the  normal  white  corpuscles  other 
cellular  elements  arc  present  in  considerable  number 
in  the  blood,  such  as  myelocytes,  this  indicates  a  definite 
blood  disease. 

If  two  diseases,  such  as  pneumonia  and  malaria,  co- 
exist in  the  patient,  the  influence  of  the  one  disease 
appears  to  overpower  the  other,  so  that  the  leucocytic 
v, uiation  of  pneumonia  only  will  be  present. 

A  variation  in  the  total  amount  of  blood  in  the  body 
no  doubt  occurs  in  certain  diseases,  but  there  is  no  simple 
practical  method  of  determining  such  variations. 

To  enumerate  the  larger  parasites,  such  as  filarial 
embryos,  no  dilution  is  required.  A  measured  quantity 
of  blood  is  taken  up  in  a  pipette  and  blown  out  on  to  a 
slide.  This  blood  is  spread  out  and  allowed  to  dry  and 
decolourized  by  placing  in  water.  The  total  number  of 
filarial  embryos  can  then  be  counted  and  reduced  to  the 
proportion  per  c.mm.  This  is  a  matter  of  no  difficulty 
and  the  only  method  to  be  relied  on.  Substitutes  that 
are  often  employed  are  to  make  a  thick  dry  blood  film  a 
certain  size,  or  to  use  cover-classes  of  a  definite  si/.e  in 
making  fluid  lilms,  or  to  make  a  certain  number  of  drops 
of  blood  in  making  the  film.    These  methods  as  substitutes 


BLOOD   COUNTS  445 

for  direct  measurement  are  inferior,  and  results  arrived  at 
by  these  methods  have  little  value.  In  the  absence  of  a 
measured  pipette  any  tine  marked  tube  may  be  used. 
The  volume  may  be  estimated  subsequently. 

The  use  of  measured  amounts  of  blood,  decolourized 
and  stained,  for  the  estimation  of  malarial  parasites  is 
practised  by  Ross.  The  results  are  reliable  with  special 
training.  The  parasites  are  distorted  during  the  process, 
but  with  practice  most  of  them  can  be  recognized.  An 
approximation  to  the  number  present  can  be  obtained 
by  first  estimating  the  number  of  leucocytes  present 
per  c.mm.,  and  then  taking  a  fluid  fresh  film  of  the 
blood  and  determining  the  relative  number  of  parasites 
to  leucocytes  in  the  film.  If,  for  instance,  ten  parasites 
are  found  in  this  film  and  100  leucocytes,  there  will  be 
one  parasite  to  ten  leucocytes,  and  if  the  number  of 
leucocytes  determined  separately  is  found  to  be  8,000 
per  c.mm.,  then  the  number  of  parasites  should  be  one- 
tenth  of  this,  or  800  per  c.mm. 

The  results  are  approximate  only,  as  leucocytes  are 
not  uniformly  distributed  in  the  fluid  film.  In  Ross' 
method  very  small  quantities  of  blood  are  taken  in  a  fine 
tube  such  as  that  of  a  clinical  thermometer  which  is 
marked.  The  volume  is  determined  by  calculation, 
but  as  these  line  tubes  are  often  not  truly  cylindrical  it 
might  be  better  to  graduate  by  the  weight  of  the  mercury 
contained  in  each  division.  Rejecting  the  first  drop 
the  measured  amount  taken  is  blown  out  on  to  a  glass 
slide  and  spread  out  and  decolourized  and  stained. 
Every  parasite  is  counted. 

By  this  method  Ross  has  been  able  to  demonstrate 
a  periodicity  in  the  increases  and  decreases  in  the 
number  of  trypanosomes  in  a  case  of  trypanosomiasis  and 
has  studied  the  variations  in  the  numbers  of  malaria 
parasites  with  special  reference  to  the  number  required 
to  produce  pyrexia  and  the  effect  of  drugs.  He 
finds  that  in  infections  with  P.  vivax  (benign  tertian) 
an     average     of      125     per     c.mm.    did      not     produce 


446  BLOOD   COUNTS 

fever,  whilst  with  P.  falciparum  (subtertian)  an 
average  cf  461  per  c.mm.  did  not  cause  pyrexia. 
He  estimates  the  usual  pyogenic  limit  in  P.  vivax  as 
between  200  and  500,  whilst  with  P.  falciparum  600  to 
1,500  might  be  adopted.  For  determination  of  the  total 
parasites  he  estimates  that  a  man  of  10  stone  will  have 
3,000,000  c.mm.  of  blood.  (For  full  particulars  vide 
"Annals  of  Tropical  Medicine  and  Parasitology,"  vol. 
iv.,  No.  1,  December  1910). 

It  is  of  some  interest  to  compare  results  obtained  by 
counting  the  leucocytes  and  parasites  in  a  fresh  fluid  film 
with  Ross's  more  accurate  method.  Douglas  Gray  in 
1901  and  Sims  in  1902,  "  Journal  of  Tropical  Medicine," 
estimated  from  their  observations  made  by  counting 
against  leucocytes  in  fluid  films  that  the  pyogenic 
limit  in  a  case  of  quartan  malaria  (P.  malarice)  was 
between  250  and  446,  and  that  in  benign  tertian  (P.  vivax) 
it  was  400  or  more. 

The  relative  numerical  proportion  of  the  parasites  to 
leucocytes,  if  determined  in  a  dry  film,  is  far  more  in- 
accurate, as  the  distribution  of  the  leucocytes  is  so  unequal 
in  such  a  film,  and  many  of  the  leucocytes  adhere  to  the 
needle,  slide  or  paper  used  in  making  the  film.  The  leuco- 
cytes in  a  thin  part  of  the  field,  such  as  is  used  for  the 
observation  of  parasites,  will  be  from  one-half  to  about 
one-tenth  of  the  proper  amount,  and  the  error  from  this 
cause  in  counting  the  parasites  will  therefore  vary  to  the 
same  extent.  An  approximation  can  also  be  obtained  by 
determining  the  average  number  of  parasites  in  a  field.  If 
the  average  number  of  red  corpuscles  in  the  same  field 
is  also  determined  the  method  is  of  value,  but  it  is 
tedious. 

In  fluids  other  than  blood,  where  parasites,  including 
bacteria,  are  numerous  and  minute,  Wright  suggests 
mixing  this  fluid  with  an  equal  quantity  of  blood  diluted 
so  that  the  number  of  corpuscles  per  c.mm.  is  known. 
The  relative  proportions  of  the  parasites  to  the  blood 
corpuscles,  as  determined  by  making  a  dried  film  of  the 


COUNTS    OF   MICRO-ORGANISMS  447 

mixture  and  staining  it,  will  then  enable  us  to  estimate 
the  number  of  organisms  present  in  any  given  quantity 
of  the  fluid. 

The  more  usual  method  of  estimating  the  number  of 
living  bacilli  in  a  fluid  is  to  take  a  measured  volume  of  the 
fluid  and  add  to  it  a  measured  quantity  of  liquefied 
gelatine  and  to  plate  it.  The  number  of  colonies  found 
in  this  plate  will  give  the  number  of  organisms  in  the 
volume  of  fluid  taken.  If  the  colonies  are  too  numerous 
the  number  found  in  any  measured  area  may  be  counted, 
and  this  result  reduced  to  terms  of  the  total  area  of  the 
plate.  If  the  number  of  organisms  is  very  great  a  second. 
or  third  dilution,  always  with  measured  amounts  of  the 
fluid  and  gelatine,  may  be  necessary. 

For  determination  of  the  number  of  bacteria  in  air, 
a  measured  amount  of  air  is  driven  through  liquefied 
gelatine,  which  is  plated,  and  the  number  of  colonies 
estimated  as  before. 

For  earth  or  solids  these  must  be  finely  divided  and 
weighed  amounts  taken. 

It  is  essential  that  the  plates  should  be  accurately 
levelled,  and  as  much  of  the  plate  as  possible  should  be 
counted.  Agar  plates  may  be  used,  and  for  complete 
investigation  incubated  at  various  temperatures.  Other 
plates  should  also  be  made  and  incubated  aerobically 
and  anaerobically. 

The  numerical  estimation  of  eggs  in  fasces  hardly 
admits  of  practical  application,  as  the  amount  of  water, 
&c,  in  the  fasces  varies  so  greatly  and  eggs  are  not  uni- 
formly distributed.  Here  loose  terms,  such  as  numerous, 
very  numerous,  moderate  number,  or  few,  are  more  in 
accordance  with  the  amount  of  information  available 
than  a  numerical  estimate  could  be,  though  that  may 
have  a  superficial  appearance  of  exactness. 

Colorimctric  Estimations  are  not  very  accurate.  They 
are,  however,  the  only  simple  methods  that  can  be  used 
with  rapidity  and  ease.  They  are  all  based  on  the  com- 
parison of  the  diluted  fluid  with  a  substance  standardized 


44* 


COl.okl.METKlC    ESTIMATIONS 


as  regards  colour.  The  principle  adopted  is  to  ^<»  arrange 
matters  that  the  equality  in  tint,  or  "matching,"  of  th<* 
two  objects  compared  is  obtained,  as  equality  in  tint  can 
be  determined  more  exactly  than  degrees  of  difference. 

In  such  colorimetric  examinations  the  same  source 
of  light  must  invariably  be  used,  and  for  general  pur- 
poses artificial,  particularly  candle  light,  is  to  be  pre- 
ferred, as  colours  seen  by  one  light  will  not  match  when 
viewed  with  another  light. 

The  methods  most  used  for  blood  work  are  Gower's 
haemoglobinometer  and  its  modifications,  in  which  gela- 
tine coloured  with  picro-carmine  is  used  as  the  standard 


Fig.  162. — Oliver's  Tintometer. 


colour  and  standardized  so  as  to  represent  a  certain 
percentage — 1  per  cent.  —  of  haemoglobin,  and  von 
Fleischl's  haanometcr  and  its  modifications,  in  which  a 
wedge  of  tinted  glass  is  used  as  a  standard,  this  being 
moved  till  the  thickness  of  the  wedge  is  such  that  the 
depth  of  colour  equalizes  that  of  a  definite  depth  of  the 
fluid  to  be  examined.  In  Oliver's  method  (tig.  162),  a 
series  of  graded  depths  of  coloured  discs  is  used  as  the 
standard.  The  colour  produced  is  compared  with  these 
standard    colours    bv  mixing  with   water   a   definite  fixed 


II.KUOdLomX 


449 


quantity  of  blood  in  a  glass  cell  of  fixed  depth  and 
capacity  placed  on  a  white  plaster  disc. 

For  all  these  methods  it  is  necessary  that  the  blood 
corpuscles  should  be  broken  up  and  the  haemoglobin 
be  in  solution.  This  can  be  done  by  dilution  with  dis- 
tilled water,  or,  better,  by  dilution  with  a  i  per  cent, 
aqueous  solution  of  carbonate  of  soda. 

In  Gower's  method  (tig.  163)  the  standard  colour  is 
equivalent  to  that  of  normal  blood  diluted  so  as  to 
represent    1   per  cent,  of  haemoglobin  dissolved  in  water. 


Fig.  163. — Gower's  H/emoglobinometer. 

The  (luid  to  be  estimated  is  placed  in  a  cylinder,  or, 
better,  in  a  flattened  tube  exactly  similar  to  that  con- 
taining the  standard,  and  is  diluted  drop  by  drop  till 
the  two  colours  are  matched.  The  tube  is  graduated  so 
that  the  degree  of  dilution  can  be  read,  and  the  amount 
of  dilution  necessary  to  produce  the  same  depth  of  colour 
indicates  the  relative  amount  of  haemoglobin  as  compared 
with  the  standard. 

In  von  Fleischl's  method  (fig.  164)  the  wedge  of 
coloured  glass  is  arranged  on  a  stand  and  illuminated 
from  below  by  a  plaster  of  Paris  disc.  Fitting  into  the 
circular  opening  of  the  stage  is  a  metal  cylinder  with  a 
glass  bottom,  and  this  cylinder  is  divided  longitudinally 
into  two  compartments.  One  is  rilled  with  diluted  and 
laked  blood  and  the  other  with  water.  Under  this  second 
compartment  is  the  wedge  of  coloured  glass,  and  this 
29 


45° 


H.KMOGLOBIN 


wedge  is  moved  horizontally  by  a  rack  and  pinion  till  the 
colour  corresponds  to  or  matches  that  of  the  diluted 
blood.  The  movement  of  the  wedge  is  indicated  on  a 
scale  graduated    by  comparison  with    haemoglobin   solu- 


Fig.  164. — von  Flkischl's  H^mometf.k. 

tions  of  varying  strength,  so  that  the  haemoglobin 
equivalent  of  the  portion  of  the  glass  wedge  in  the  held 
can  be  read  at  once. 

Tallquist's  Hcvmoglobin  Scale. — This  is  a  scale  of  colours 
representing  the  shades  of  red  corresponding  to  definite 
percentages  of  haemoglobin.  A  book  of  filter  papers 
accompanies  the  scale. 

To  estimate  the  haemoglobin  a  drop  of  blood  is 
absorbed  by  the  filter  paper,  and  when  the  gloss  has 
passed  off  this  is  compared  with  the  scale — the  number 
opposite  the  shade  of  colour  which  most  nearly  corre- 
sponds to  it  gives  the  percentage  of  haemoglobin.  The 
estimation  should  be  done  by  daylight.  It  is  not  very 
accurate,  but  the  results  are  about  as  reliable  with  this 
method  as  with  any  other  in  common  use.  It  has  the 
great  advantages  of  simplicity  and  quickness  of  perform- 
ance. 


45 r 


CHAPTER   XXIV. 

Statistics. 

Statistics. — Statistics  as  reliable  as  those  obtainable  in 
England  can  rarely  be  obtained  in  the  Tropics,  and  an 
important  source  of  information  is  thus  wanting.  Even 
statistics  of  births,  deaths,  and  the  more  important 
diseases,  both  of  Europeans  and  natives,  have  to  be 
admitted  with  great  caution,  and  local  knowledge  of  the 
manner  in  which  they  are  compiled  is  essential  before 
giving  them  even  the  slightest  consideration.  With  local 
knowledge  the  statistics  may  be  very  valuable,  but  -even 
then  the  errors  due  to  selection  of  statistics,  a  personal 
equation,  greatly  reduces  their  value. 

Statistics  for  tropical  work  therefore  must,  to  a  great 
extent,  be  the  result  of  individual  work,  so  that  the  large 
numbers  that  European  statisticians  are  accustomed  to 
work  with  are  not  obtainable. 

It  is  therefore  of  the  greatest  importance  at  the  outset 
that  the  probable  error  due  to  working  with  small  figures 
should  be  clearly  grasped. 

The  best  method  of  arriving  at  the  probable  error  in 
cases  where  a  series  of  facts  is  divided  into  two  groups 
is  by  Poisson's  formula  : — 

If  g  =  the  total  number  of  cases, 
m  =  the  number  in  one  group, 
And  n  =  the  number  in  the  other  group,  so  that  m  +  n  =  g, 

the  proportion  of    each   group  to  the  whole  series  will 

be  respectively  -  and  -  ;   but  these  proportions  will  vary 

in    succeeding    series,    and   the    extent    of    the   variation 


452  POISSON  S    FORMULA 

will  be  within  the  limits  represented  by  the  following 
expression  : — 

'"      _1_     ->      v'2_mn_ 
8      —     ~  g* 

Obviously,   the  larger   the  value   of  g  the  less   will  be 

the  value  of  2   *  — '-,    and    consequentlv  the   less    will    be 

g"  1 

the  limits  of  error  in  the  simple  proportion,  ™,  and  con- 
versely the  smaller  the  value  of  g  the  greater  will  be 
the  limits  of  error. 

For  example,  if  in  100  cases  of  beri-beri  ten  have 
been  fatal,  and  it  is  required  to  determine  the  limits 
of  error  in  assuming  this  proportion  to  hold  for  the 
next  10,000  cases  : — 

Applying  the  formula.     Here  g  =  100,  m  =  10,  n  =  90. 

10         1  V '2  X   10  X  go 

100      —     ""  100  X   100  X   100 

=     i     ±  2   X    -0428  =    70    ±   -0856 
•0856  represents  the  variations  per  unit. 

So  that  in  the  next  10,000  cases — 

There  may  be  1,000  -f-  856  =  1,856  deaths  ; 
Or  1,000  —  856  =     144  deaths. 

This  formula  only  deals  with  the  mathematical  relation- 
ship of  the  figures.  Further  allowance  has  to  be  made 
for  errors  of  observations  and  the  numerous  uncertain 
factors  met  with  in  any  statistics. 

A  mere  consideration  of  the  effects  of  the  extent  of 
the  possible  mathematical  error  in  dealing  with  small 
figures  suffices  to  indicate  the  care  that  is  necessary 
and  the  multitude  of  observations  that  are  requisite 
before  formulating  a  definite  conclusion.  In  spite  of  the 
magnitude  of  this  error,  of  the  numerous  possibilities, 
of  errors  of  observation,  and  even  of  the  fact  that  mere 
increase  in  the  number  of  observations  may  only  multiply 
the  same  error  or  doubtful  point,  as  the  same  source  of 
error  may  be  included  in  each  observation,  the  acquisi- 
tion and  use  of  statistics  is  of  high  value,  and  often 
indicates  a  correct  conclusion.  The  liability  to  error 
diminishes  but  does  not  destroy  this  value. 


EVIDENCE  453 

It  is  customary  to  indicate  all  results  in  percentages, 
and  no  doubt  this  method  renders  comparison  easy  ;  but 
a  consideration  of  the  formula  of  probable  error  shows 
that  i  in  4  is  by  no  means  the  same  thing  as  25  in  100. 
The  number  of  observations  made  must  be  included  in 
any  account,  and  whenever  possible  these  observations 
should  greatly  exceed  the  100.  The  consideration  of 
the  magnitude  of  the  probable  mathematical  error  under 
the  most  favourable  circumstances  should  lead  to  as 
great  an  exactitude  as  possible  and  avoidance  of  other 
and  avoidable  sources  of  error. 

Value  of  Evidence.  —  Considerable  judgment  as  well 
as  caution  is  requisite  in  obtaining  information  other 
than  that  derived  from  personal  observation. 

As  regards  occurrence  of  diseases,  parasites,  &c,  much 
of  the  information  received  must  be  taken  with  great 
caution,  as  it  is  often  from  laymen  and  untrained  ob- 
servers. Even  more  in  the  Tropics  than  in  England 
such  persons  hold  theories  either  of  their  own  or  derived 
from  others,  and  are  anxious  to  bring  forward  only  facts 
which  are  in  support  of  these  theories. 

It  is  well  in  making  enquiries  to  be  careful  to  limit 
the  enquiry  to  points  that  are  within  the  power  of  any 
ordinary  observer.  It  is  not  well  to  discard  altogether 
such  evidence,  as  on  many  important  points  information 
can  be  derived,  and  in  some  of  these  the  liability  to 
error  is  no  greater  than  with  a  professional  observer. 

Various  points  in  connection  with  malaria  might  be 
well  taken  as  illustrations,  both  of  the  value  of  such  infor- 
mation and  the  errors  that  are  likely  to  occur  as  a  result 
of  too  much  confidence  in  such  information,  as  well  as 
of  the  general  methods  which  have  been  adopted  deter- 
mining etiological  and  other  factors. 

These  points  comprise:  (1)  As  regards  Individuals. — 
Their  susceptibility  to  the  disease  and  the  effects  of  the 
disease,  including  liability  to  relapses,  length  of  period 
of  intermission  between  relapses,  and  any  evidence  of 
the  acquirement  of  immunity. 


454  STATISTICS   OF   MALARIA 

(2)  As  regards  the  Population  in  General. — Suscepti- 
bility, and  any  factors,  age,  race,  or  habits  influencing  it. 
Mortality    per     1,000    of   the  population    at    various   age 

periods;  and  case  mortality  in  treated  and  untreated 
cases  ;  liability  to  any  special,  immediate  or  remote 
complications  ;  effect  on  general  health  ;  any  evidence 
of  acquisition  of  immunity. 

(3)  As  regards  the  Place. — This  should  include  enquiries 
as  to  any  special  house,  village  or  district,  as  well  as 
the  country  in  general,  where  the  disease  is  more  or 
less  prevalent  than  the  average.  Seasonal  variations 
and  their  effects,  particularly  rainfall,  temperature,  and 
any  cause  affecting  level  of  subsoil  water.  Any  facts 
known  as  to  the  prevalence  of  the  known  main  factor — 
in  the  case  of  malaria,  prevalence  of  Anophelince—'m  the 
spread  of  the  disease.  Some  numerical  estimate,  endemic 
index,  of  the  liability  to  infection. 

Most  of  these  points  can  be  determined  to  some  extent 
by  careful  enquiries,  though  the  results  must  be  con- 
firmed by  observation,  or  where  possible  by  the  adoption, 
as  a  check,  of  other  methods. 

The  results  obtained  in  this  way,  though  not  to  be 
implicitly  relied  upon,  will  be  a  valuable  guide  to  the 
direction  of  researches  required  in  a  district  or  country. 

Liability  to  Infection.  — ■  Enquiries  as  to  individuals 
necessitates  a  selection  of  cases,  and  information  of  a 
reliable  nature  can  onlv  be  obtained  on  every  point 
from  few  persons.  In  the  case  of  newcomers  the  date 
of  arrival  in  a  country  and  the  subsequent  movements, 
with  approximate  dates,  are  usually  to  be  trusted.  The 
date  of  the  first  attack  of  malaria  can  generally  be 
obtained.  Sufficient  information  about  the  attack,  such 
as  the  character  and  duration  of  the  "fever";  the 
effect  of  quinine,  and  absence  of  any  other  cause  of 
pyrexia,  such  as  septic  infection  or  pneumonia,  must  be 
ascertained  to  render  it  probable  that  the  attack  was 
malarial. 

Any  form  of    indisposition    in    the    older    residents   is 


RELAPSES  455 

so  frequently  called  malaria  that  less  reliance  is  to  be 
placed  on  these  than  on  the  history  of   the  first  attack. 

In  malaria  it  must  always  be  remembered  that  relapses 
are  so  common  that  a  second  attack,  even  at  an  interval 
of  several  months,  does  not  prove  a  second  infection. 

The  Liability  to  Relapses  is  more  difficult  to  determine, 
but  with  a  fair  number  of  individuals  it  can  be  ascer- 
tained, and  great  individual  variations  will  be  found. 
In  newcomers  three  weeks  to  a  month  is  a  common 
interval,  whilst  in  others  the  period  may  be  as  long  as 
four  or  six  months.  In  this  connection  careful  enquiries 
as  to  the  habits  as  regards  quinine  are  of  great  import- 
ance, as  if  quinine  is  taken  constantly,  even  in  small 
doses,  the  relapse  is  often  postponed  till  the  quinine  is 
discontinued. 

Increase  in  the  Interval  between  Relapses. — Any  obser- 
vations as  to  increase  in  the  interval  between  the  relapses 
with  increased  length  of  residence,  or  diminution  in  the 
severity  of  the  attacks,  may  indicate  that  a  degree  of 
immunity  has  been  acquired,  and  the  length  of  residence 
required  for  this  is,  to  a  great  extent,  an  individual 
peculiarity,  though  shorter  in  all  in  the  more  malarial 
districts. 

As  regards  the  population  in  general,  it  is  essential 
that  the  actual  numbers  of  the  different  races  represented 
be  known  before  any  use  can  be  made  of  totals,  such 
as  number  of  deaths,  admission  to  hospital,  &c.  This 
warning  may  appear  superfluous,  but  it  is  not.  In  pub- 
lished reports  one  of  the  commonest  errors  is  to  speak 
of  a  disease  as  being  more  or  less  prevalent  in  a  district 
on  the  ground  of  the  number  of  cases  seen,  not  as  it 
should  be,  on  the  proportion  of  the  susceptible  population 
attacked. 

It  is  in  connection  with  blackwater  fever  and  yellow 
fever,  in  its  diagnosable  form,  that  such  errors  are  most 
common. 

Age  Incidence. — Personal  observations  should  be  made 
on  unselected  cases  and  the  number  of  cases  examined 


45^  MORTALITY 

mentioned    in    the    tabic,    with    the    percentages.     Ages 

cannot  be  ascertained  with  certainty,  especially  in  coun- 
tries where  the  differences  in  season  are  not  very  marked. 
With  children  age  has  to  he  estimated  from  the  size, 
teeth  *  and  development.  In  adults  knowledge  of  local 
history  and  notable  events,  the  dates  of  which  can  be 
fixed,  are  of  considerable  value.  Age  periods  of  five 
years  are  usually  taken,  but  it  is  of  the  utmost  import- 
ance in  malarial  investigation  to  subdivide  the  first 
quinquennial  period  and  further  subdivide  the  first  year 
into  quarters.  The  first  quarter  should  be  subdivided 
into  months.  Malaria  is  rare  till  the  end  of  the  first 
month. 

As  an  age  period  the  first  ten  years  should  never  be  taken 
as  a  whole,  as  such  different  results  are  obtained  in  a 
village,  or  in  a  series  of  observations,  if  a  large  proportion 
are,  say,  under  four,  or  only  a  small  proportion.  Conclu- 
sions drawn  from  the  incidence  of  malaria  in  the  first 
ten  years  of  life,  taken  as  a  whole,  are  often  misleading. 

Mortality  is    best   estimated    at   the    rate    of    so    many 

*  Ages  at  which  teeth  are  cut  in  Europeans.  The  differences  in 
native  races  have  not  yet  been  worked  out.  Table  kindly  supplied 
to  us  by  Mr.  K.  W.  Goadby. 

Temporary  Dentition. 


Central  incisors 

5th 

to    8th  month. 

Lateral  incisors 

7th 

to  10th         ,, 

First  molars    ... 

1 2  th 

to  14th         „ 

Canines 

14th 

to  20th          ., 

Second  molars 

20th 

to  30th         ,, 

Permanent 

Dentition. 

Upper 

Jaw. 

Tower  Jaw. 

Central  incisors 

77  years 

7  years, 

Lateral  incisors 

8 

11 

-       8       „ 

Canines  ... 

..,     1 1 

)• 

...     10      „ 

Premolar  I. 

]  o 

') 

10      ,, 

Premolar  II.     ... 

ii 

11 

...     11       „ 

Molar  I 

...       b\ 

)j 

•••       7       „ 

Molar  1 1 

12 

11 

...      12       „ 

Molar  III 

...      24 

11 

...     24       „ 

MORTALITY  457 

deaths  per  1,000  per  year,  as  then  the  results  can  he 
compared.  If  dealing  with  short  periods,  as,  for  instance, 
one  week,  the  death-rate  would  be  the  proportion  of 
deaths  per  1,000  of  the  population  in  that  period, 
multiplied  by  52.  If  a  long  period,  say  ten  years,  is 
taken,  the  death-rate  would  then  be  represented  by  the 
number  of  deaths  per  1,000  divided  by  10. 

The  factors  necessary  are  the  number  of  persons  of 
the  required  class  alive  at  the  commencement  of  the 
period,  the  number  of  deaths  of  this  class  who  died 
from  the  disease  which  it  is  desired  to  investigate  in 
the  period,  and  the  length  of  the  period. 

Case  Mortality  is  the  percentage  representing  the  pro- 
portion of  cases  terminating  fatally.  The  number  of 
cases  of  the  disease  and  the  number  of  deaths  from  the 
disease  are  the  only  two  factors  requisite.  If  it  is  desired 
to  compare  the  "  case  mortality "  in  different  years  or 
other  periods  of  time,  cases  occurring  in  those  periods 
only  must  be  included.  In  malaria  untreated  and  treated 
cases  must  be  considered  separately,  and  the  treatment 
mentioned  as  the  case  mortality  is  so  much  reduced 
by  effective  treatment.  In  yellow  fever  not  only  must 
the  races  be  kept  distinct,  but  the  period  when  they 
were  last  exposed  to  a  definite  epidemic  must  be  clearly 
indicated. 

Remote  or  Indirect  Mortality  is  the  mortality  due  to 
remote  complications,  visceral  changes  and  increased 
liability  to  other  diseases,  or  to  the  tendency  which 
malaria  appears  to  have  to  aggravate  some  diseases. 
Our  knowledge  of  this  branch  of  the  subject  is  most 
inaccurate  and  requires  complete  revision. 

The  effect  on  general  health  varies  greatly  in  different 
conditions,  and  under  circumstances  little  understood. 
Splenic  enlargement,  anaemia  and  diminished  rate  of 
growth  are  the  most  definite.  Susceptibility  to  tuber- 
culosis appears  to  be  induced  by  chronic  malaria  in 
countries  where  tuberculosis  is  prevalent.  The  effect 
on    the   general    health,    apart    from    the    actual    attacks, 


45  <s  IMMUNITY 

whether  mild  or  pernicious,  varies  according  to  race. 
Some  races,  as  children,  have  an  uncertain  degree  of 
"  tolerance,"  at  least  as  regards  toxic  effects. 

Period  of  natural  incubation  and  its  variations  can 
be  determined  from  the  histories  of  patients,  and  then 
inquiry  must  be  limited  either  to  first  attacks  or  to  other 
attacks  in  which  a  long  interval  has  elapsed.  The  most 
common  history  given  is  of  some  immediate  antecedent. 
Exposure  to  chill,  constipation,  change  of  residence,  par- 
ticularly from  a  warmer  to  a  cooler  place,  and  even 
cessation  of  travelling,  are  given  as  the  causes  of  the 
attack.  These  causes  are  not  to  be  taken  as  those  of 
the  infection,  though  they  may  determine  or  accelerate 
the  manifestation  of  the  disease. 

The  time  of  actual  onset  of  symptoms  can  usually 
be  told  with  certainty,  but  the  time  of  infection  is  diffi- 
cult to  determine.  The  frequency  with  which  travelling 
in  one  form  or  other  enters  into  the  causation  is  usually 
to  be  ascribed  to  passing  through  a  highly  malarial 
district,  or  even  to  spending  some  hours  in  a  house 
where  infected  mosquitoes  are  to  be  found.  With  a 
sufficient  number  of  cases  it  is  sometimes  easy,  as  in  the 
case  of  a  steamer,  or  in  persons  travelling  over  known 
routes,  to  fix  on  the  date  of  infection  as  the  date  on 
which  a  halt  was  made  at  a  notoriously  malarial  place. 
Such  cases  show  the  wide  limits  of  the  period  of  natural 
incubation,  often  longer  than  those  which  have  been 
determined  experimentally  by  feeding  infected  mosquitoes 
on  susceptible  persons. 

The  evidence  of  immunity  is  to  be  considered  under 
two  heads  :  (i)  Age  incidence  of  the  disease  in  natives 
and  cessation  of  attacks  with  advancing  years.  (2)  In 
newcomers  the  residential  period  during  which  attacks 
occur,  and  any  evidence,  by  the  diminishing  frequency 
or  severity  of  attacks,  that  some  immunity  is  acquired. 
Immunity  from  a  disease  must  be  clearly  distinguished 
from  "tolerance"  or  immunity  from  the  effects  of  the 
invasion  by  the  parasites. 


SEASONAL   VARIATION  459 

With  malaria  it  is  important  to  consider  whether 
there  are  periods  in  which  from  climatic  conditions 
infections  do  not  take  place.  In  the  case  of  individuals, 
ii  there  are  periods  during  which  they  are  not  resident 
in  places  where  malarial  infection  is  possible.  Immunity 
is  destroyed  or  diminished  by  such  periods,  so  that  if 
they  are  long  immunity  is  not  acquired  at  so  early  a 
period,  or  at  all.  There  is  evidence  that  immunity  is 
not  of  long  duration  in  malaria,  but  more  exact  observa- 
tions are  required  on  this  point. 

In  any  consideration  of  immunity  the  liability  to 
infection — endemic  index — must  be  taken  into  account, 
as  with  a  low  endemic  index  individuals  only,  not  a 
class,  will  acquire  immunity. 

(3)  As  regards  Place. — In  considering  any  place  it  is 
important  to  bear  in  mind  that  malaria  is  a  local  disease, 
and  that  even  in  houses  close  together  one  will  be  more 
malarial  than  another.  Still  more  so  are  different  quarters 
of  the  same  town  or  district,  and  the  localities  where  the 
disease  is  most  prevalent  vary  from  year  to  year.  These 
differences,  and  the  causation  of  the  variation  in  the 
differences,  require  local  investigation  in  all  cases. 

Seasonal  variation  may  act  in  two  ways,  first  by  ren- 
dering the  conditions  more  favourable  for  the  multipli- 
cation of  Auoplietiua,y  and  secondlv  by  presenting  condi- 
tions more  favourable  for  the  development  of  the  malaria 
parasites  in  the  mosquitoes.  Rainfall,  both  the  amount 
and  distribution,  i.e.,  whether  in  frequent  light  showers 
with  short  intervals,  or  heavy  downpours  with  long 
intervals,  is  of  great  importance,  but  the  effect  may  vary 
with  the  same  monthly  rainfall,  as  occasional  heavy 
showers  flush  out  and  destrov  mosquito  larvae,  whilst  the 
same  amount  of  rain  falling  slowly  will  merely  increase 
the  size  and  maintain  the  same  breeding  places.  The 
level  of  the  subsoil  water  may  be  more  affected  by  distant 
rain  than  by  the  local  rainfall,  and  thus  distant  rainfall 
may  be  a  cause  of  the  unhealthiness  of  a  place.  Rain  on 
mountains  or  hills  behind  a  station  is  an  example  of  this. 


4<>0  ENDEMIC    INDEX 

Where  there  .ire  snow-covered  mountains,  as  in  Equa- 
torial Africa,  the  water  supply  is  dependent  on  the  melt- 
ing of  this  snow,  and  therefore  a  high  temperature 
increases  the  water  supply  of  a  large  district      A   high 

temperature  within  certain  limits  causes  more  ra  )id 
breeding  of  mosquitoes,  causes  them  to  require  food  and 
therefore  to  attack  men  more  frequently,  and  is  favourable 
to  the  rapid  development  of  the  malaria  parasites,  and  so 
in  all  these  ways  will  favour  the  spread  of  malaria. 

The  specie's  of  Anophelince  present  and  of  those  most 
numerous  in  the  district  should  be  determined,  and 
these  species  of  mosquitoes  should  be  tested  as  to  the 
readiness  with  which  they  may  become  infected  by  the 
malaria  parasite.  Different  species  vary  greatly  in  this 
respect,  and  even  with  the  same  species  infection  seem> 
to  occur  with  varying  difficulty  under  different  circum- 
stances. As  a  rule  mosquitoes  reared  from  larvae  are 
not  as  easily  infected  as  those  of  the  same  species  caught 
in  the  adult  stage.  The  possible  or  known  circumstances 
affecting  the  development  of  the  parasites  are  the  tem- 
perature, the  age  of  the  mosquito,  whether  impregnated 
or  not,  and  the  nature  of  the  food  previously  taken  by  the 
mosquito,  and  probably  other  conditions,  which  all  require 
local  investigation. 

Endemic  Index. — A  numerical  estimate  of  the  liability 
to  malarial  infection  is  an  important  factor  to  deter- 
mine. The  number  of  malarial  attacks,  or  of  hospital 
admissions  for  malaria,  from  a  known  number  of  persons, 
is  of  little  value,  even  if  the  diagnosis  is  confirmed  in 
every  case  by  blood  examinations,  as  these  admissions 
will  include  recurrences  and  relapses,  which  will  vitiate 
the  figures,  eg.,  a  man  infected  once  with  malaria  may 
have  a  dozen  attacks  of  malaria  as  a  result  of  this  single 
infection  in  a  year,  or  he  may  have  only  one.  In  the 
first  instance  he  would  appear  in  returns  as  12,  in  the 
second  as  1,  though  in  both  instances  for  our  purpose 
his  infection  should  be  represented  as  1. 

If    first   attacks   only   are   included    this   difficulty    does 


ENDEMIC    INDEX  46 1 

not  occur,  and  first  attacks  have  the  further  advantage 
of  being  usually  severe,  and  in  persons  who  have  not 
yet  acquired  the  habit  of  treating  themselves.  These 
therefore  usually  come  under  medical  observation. 

For  an  estimate  of  the  liability  to  infection,  or  endemic 
index,  by  this  method,  the  factors  to  ascertain  are  the 
dates  of  first  attacks  of  malaria  occurring  during  the 
course  of  the  observations,  verified  by  blood  examina- 
tions or  in  other  ways,  effect  of  quinine,  &c,  and  the 
length  of  residence  previous  to  the  attack,  and  the 
number  of  newcomers  who  have  escaped  infection 
during  the  period.  As  a  separate  estimate  a  statement 
by  as  large  a  proportion  as  possible  of  the  resident 
population  as  to  the  length  of  time  they  had  each 
resided  in  the  country  before  their  first  attack  of  malaria. 
These  figures  usually  lead  to  much  the  same  result. 
Reliance  has  to  be  placed  on  histories  only,  and  errors 
may  occur,  though  each  factor  is  one  which  most  of  the 
residents  are  capable  of  observing. 

By  this  method  the  length  of  residence  in  weeks  or 
months  that  is  ordinarily  required  for  an  attack  of 
malaria  is  determined.  The  period  of  incubation  we 
know  varies,  but  is  commonly  from  ten  days  to  three 
weeks,  and  this  period  should  be  subtracted  from  the 
length  of  residence  required  for  an  attack  of  malaria 
to  develop  in  order  to  obtain  the  period  of  residence 
required  for  infection. 

Where  bodies  of  men  are  working  together  and  are 
under  medical  observation,  as  in  regiments,  gangs  of 
workmen,  &c,  this  method  is,  we  believe,  the  best  and 
simplest,  and  includes  no  sources  of  error  that  are  not 
common  to  other  methods. 

In  such  an  estimate  all  persons  who  were  born  and 
have  lived  in  malarial  countries  for  prolonged  periods 
should  be  excluded  ;  also  those  who  have  contracted 
malaria  in  other  malarial  countries.  For  these  exclu- 
sions there  are  two  reasons — (1)  to  avoid  including 
relapses,  and  (2)  to  avoid  including  persons  who  may  be 
immune. 


462  ENDEMIC    INDEX 

A  somewhat  similar  method  is  to  determine  the  pro- 
portion of  untreated  natives  who  harbour  the  parasites 
of  malaria.  In  this  method  the  ages  must  he  known, 
and  unselected  children,  including  those  apparently  in 
good  health,  must  be  examined.  Children  should  form 
a  large  proportion  of  the  eases. 

This  method  has  been  extensively  used,  and  an  arbitrary 
standard,  ten  years,  has  been  seleeted  ;  the  proportion  of 
children  under  10  years  of  age  with  malarial  parasites  is 
then  taken  as  the  index. 

A  more  satisfactory  method  is  to  determine  the  propor- 
tion at  different  ages.  Thus  in  one  district,  whilst  86 
per  cent,  of  the  children  under  2  years  of  age  were  in- 
fected with  parasites  of  malaria,  only  28  per  cent,  of  those 
from  5  to  10  harboured  them.  If,  therefore,  in  such  a 
place  most  of  the  examinations  were  made  in  young 
children,  a  much  higher  index  would  be  obtained  than 
if  most  of  the  children  were  over  5. 

In  many  of  the  determinations  no  further  information 
than  "children  under  10  years  of  age "  has  been  given 
and  in  some  of  them  the  number  of  children  examined 
is  very  small. 

It  is  not  very  easy  in  some  places  to  get  a  sufficient 
number  of  children  for  examination,  but  with  patience 
it  can  generally  be  done.  As  these  cases  are  untreated, 
many  of  them,  if  not  most,  will  have  had  the  parasites 
for  considerable  periods,  and  therefore  the  figures  only 
indicate  antecedent,  perhaps  remote,  infection.  If  young 
children  were  examined  monthly  till  parasites  were  found, 
the  liability  to  infection  under  native  conditions  would 
be  determined  more  accurately.  In  making  any  series  of 
blood  examinations  for  such  purposes  the  time  selected 
should  be  during  a  period  of  settled  weather.  If  examina- 
tions are  made  during  a  change,  particularly  from  hot  to 
cold,  the  parasites  will  be  more  easily  found,  as  the  effect 
of  chill  is  to  favour  the  development  of  the  parasites. 
Examinations  made  at  such  times  will  therefore  show  a 
higher  index  than  those  made  in  settled  weather. 


SPLEEN   TEST  4O3 

The  Spleen  Test,  or  the  proportion  of  persons  with 
enlarged  spleens,  is  useful  if  age  and  race  are  taken  into 
account.  It  is  of  more  value  amongst  negroes  than 
amongst  other  races,  as  the  negro  spleen  does  not  con- 
tinue to  enlarge  after  immunity  has  been  acquired  in  the 
same  way  that  the  spleens  of  many  individuals  of  other 
races  do.  The  test  can  be  used  easily,  as  there  is  nothing 
in  the  examination  to  excite  alarm  or  frighten  the 
children,  and  can  be  made  more  quickly  than  any  other 
examination. 

It  indicates  only  antecedent,  probably  remote,  infec- 
tion, and  is  less  certain  proof  of  antecedent  infection 
than  the  presence  of  parasites. 

A  large  proportion  with  enlarged  spleens,  between  2  and 
5  years  of  age,  is  an  indication  of  a  high  endemic  index. 
If  the  presence  of  malaria  in  a  district  is  proved,  the 
absence  of  enlarged  spleens  in  negro  adults,  or  a  low 
proportion  between  10  and  15,  is  equally  a  proof  of  a 
high  endemic  index,  whilst  if  the  proportion  of  enlarged 
spleens  in  adult  negroes  is  appreciable  or  large  in  those 
between  10  and  15  the  endemic  index  is  low.* 

The  determinations  obtained  by  the  spleen  test  are 
less  liable  to  be  influenced  by  meteorological  conditions 
than  the  test  by  blood  examinations  ;  they  are  easier,  and 
can  be  made  in  a  larger  number  of  cases,  but  otherwise 
are  less  accurate,  as  the  conditions  that  lead  to  splenic 
enlargement  after  malarial  infection  vary  and  are  not 
thoroughly  understood,  and  splenic  enlargement  in  a 
varying  proportion  is  due  to  other  causes. 

Another  proposed  method  of  estimation  of  the  index 
is  by  determining  the  proportion  of  the  Anophelince  that 
are  found  to  be  infected  with  the  parasites  of  malaria. 
For  this  method  to  be  of  value  the  mosquitoes  must  be 
selected  from  different  houses  and  places  in  equal  pro- 
portion, as  it  will  be  found  that  there  are  great  variations 


*  With  no  other  race  but  the   Negro  can   such    conclusions    be 
drawn  with  certainty.     ( Vide  page  23.) 


4'>4  SPLEEN   TEST 

m  this  proportion  in  adjoining  houses  and  at  different 
times.  One  good  "crescent  case1'  will  infect  almost 
every  Anopheline  of  certain  species  that  bites  the  patient, 

whilst  only  a  small  proportion  of  those  that  bite  the 
more  numerous  poor  crescent  cases  will  be  infected. 
Anophelince  in  European  houses  are  rarely  found  to  be 
infected,  whilst  in  an  overcrowded  native  house,  where 
there  is  no  protection  of  the  inmates  from  mosquitoes, 
or  in  a  hospital,  a  large  proportion  of  infected  Anophelines 
may  be  found. 

The  proportion  of  infected  mosquitoes  is  not  the  real 
test  so  much  as  the  number  of  infected  mosquitoes,  so 
that  in  these  estimates  the  number  of  Anophelince  that 
bite  a  man  per  hour  or  each  day  is  also  required. 

A  high  endemic  index,  as  determined  by  the  other 
methods,  will  be  found  in  places  were  Anophelince  are 
very  numerous,  even  when  the  proportion  infected  is 
very  small. 

It  must  always  be  remembered  that  a  place  with  a  large 
number  of  Anophelince  of  species  known  to  be  efficient 
carriers  of  the  parasite,  even  if  free  from  malaria  at  any 
one  time,  has  the  potentialities  of  a  high  "endemic 
index  "  if  the  place  be  occupied  by  newcomers  or  other 
persons  susceptible  to  malarial  infection.  This  is  the 
reason  that  railway  and  engineering  works  are  so  often 
attended  with  outbreaks  of  malaria,  even  when  con- 
ducted in  places  that  previously  were  not  considered  to 
be  very  malarious.  Anophelince  are  present  and  perhaps 
numerous  ;  in  any  case  many  new  breeding-places  are 
formed  during  excavations,  and  the  mosquitoes  become 
numerous.  Gangs  of  workmen  are  crowded  together 
in  temporary  huts,  and  they  are  not  protected  from 
mosquito  bites.  The  workmen  will  include  susceptible 
newcomers,  and  frequently  some  persons  harbouring 
parasites.  A  single  good  "  crescent  case,"  often  a  man 
with  no  symptoms  of  malaria,  will  infect  a  number  of 
mosquitoes,  and  in  the  course  of  some  ten  days  or  so 
these  mosquitoes  will  infect  any  susceptible  persons  who 


ENDEMIC   INDEX  465 

sleep  in  the  hut,  and  these  persons  will  in  about  twelve 
davs  develop  an  attack  of  malarial  fever. 

The  earliest  numerical  estimate  was  arrived  at  by 
determining  the  proportion  of  persons  at  different  ages 
whose  organs  contained  malaria  pigment.  This  method 
can  only  be  adopted  under  circumstances  where  post- 
mortem examinations  can  be  obtained  in  both  children 
and  adults.  The  results  indicate  antecedent  malaria 
infection.  The  method,  though  fairly  good,  is  only  of 
limited  value,  as  the  large  number  of  post-mortem-  examina- 
tions required  can  be  obtained  in  few  places. 

These,  then,  are  the  main  methods  for  the  determina- 
tion of  the  endemic  index  : — 

(1)  By  determining  the  length  of  residence  required 
to  render  malarial  infection  probable  in  susceptible  new- 
comers. 

(2)  The  ages  at  which  the  largest  proportion  of  natives 
harbour  the  parasites  of  malaria. 

(3)  Ages  at  which  splenic  enlargement  is  common. 

(4)  Percentage  of  persons  dying  from  all  causes  with 
malarial  pigmentation  of  the  organs. 

(5)  Number  of  infected  Anophelince. 

Other  evidences,  though  too  complicated  by  other 
factors  to  be  used  numerically,  are  a  high  infantile  death- 
rate  amongst  the  natives,  particularly  a  high  death- 
rate  from  convulsions  in  infants  over  six  months  ;  a 
high  European  death-rate  not  due  to  other  endemic 
diseases,  such  as  yellow  fever  or  cholera ;  and,  we  are 
inclined  to  add,  the  occurrence  of  blackwater  fever. 

Graphic  representations  in  the  form  of  "charts"  are 
useful  as  indicating  the  main  results  of  any  enquiry,  as 
they  are  easier  to  follow  with  the  eye  than  columns  of 
figures  or  rows  of  statistics. 

The  essential  of  a  good  chart  is  that  it  should  be 
capable  of  translation  back  into  figures,  i.e.,  a  chart 
should  be  such  that  it  can  be  read. 

The  principle  of  charting  on  a  plane  surface  in  two 
dimensions  is    that    the    horizontal   line    represents    one 

30 


466 


CHARTS 


factor,  usually  time  or  periods  of  time,  whilst  the  vertical 

represents  the  other  factor. 

Bach  of  the  factors  should  be  represented  according 
to  scale  in  order  that  it  can  be  read.  This  point  is 
often  overlooked,  even  in  the  familiar  temperature  charts, 
in  that  whilst  the  height  of  the  temperature  is  recorded 
correctly  in  the  vertical  columns,  the  distances  measured 
horizontally  between  the  points  representing  the  different 
observations  are  equally  spaced,  so  as  to  look  neat,  whilst 
the  real  intervals  of  time  are  irregular,  e.g.,  temperatures 
taken  at  2,  10,  12,  3  should  not  be  recorded   on  a  four- 

CHART  I. 

Negroes  (Native  Africans).— Hausa  and  Yomba  Children, 

320;     Hausa     Adults,     100.       Compiled     from     Official 
Report,  Lagos,  of  W.  H.  G.  H.  Best. 


Age    in 

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hour  chart,  as  if  the  time  intervals  were  equal,  but  should 
be  so  recorded  that  the  distances  measured  on  the  hori- 
zontal line  are  unequal  in  the  proper  proportion. 

In  that  case  the  chart  can  be  correctly  translated  back 
into  figures,  otherwise,  if  represented  as  equi-distant,  tin- 
translation  would  read  4,  8,  12,  4. 

The  limit  to  the  translation  of  the  chart  is  the  scale 
of  the  chart  ;  where  the  intervals  allowed  for  time  are 
small,  translation  can  only  be  approximate. 

In  blood  charts  it  is  usual  to  represent  the  heights  as 
percentages  of  normal,  as  real  figures  would  require  such 


CHARTS 


467 


enormous  charts  if  it  were  desired  to  represent  graphi- 
cally on  the  same  scale  both  red  and  white  corpuscles. 
In  such  cases  it  is  better  to  keep  to  the  figures  or  to  use 
different  vertical  scales  for  the  more  numerous  and  more 
scanty  elements.  It  must  be  clearly  indicated  what  each 
line  on  the  chart  represents. 

Too  many  lines  on  one  chart  are  difficult  to  follow, 
and  the  only  cases  in  which  it  is  advisable  to  have  two 
or  more  lines  is  when  it  is  desired  to  compare  two  or 
more  results. 

CHART    II. 

Negroes  (Native  Africans),    Central   Africa. — 714   Native 

Children   under  15,  and  numerous  Adults. 


Ago  in 
Years. 

2     5        10       15       20      25      30      35      40              50 

1       00 

%o 

H 

^co 

^,50 

sJ  30 

g^  20 

4  »* 

r 

The  different  methods  of  determining  the  endemic 
index  of  malaria  are  conveniently  rendered  graphically 
and  serve  as  illustrations  of  the  method.  Chart  I.  is 
compiled  from  an  official  report  of  W.  H.  G.  H.  Best,  of 
the  Lagos  Medical  Service,  and  formerly  of  the  London 
School  of  Tropical  Medicine,  which  is  the  earliest  report 
published  that  gives  sufficient  details  for  the  determination 
of  the  age  incidence. 

No  cases  are  given  under  3  months  of  age,  and  those 
under  6  months  are  very  few.  The  chart  shows  clearlv 
that  under  the  conditions  of  a  native  life  a  large  propor- 
tion of  children  are  infected  in  less  than  six  months, 
and  practically  all  in  less  than  a  year,  whilst  the  number 


468 


CHARTS 


of  infected  children  after  live  years  is  so  small  that   the 

majority  must  have  acquired  immunity. 
Chart  II.  shows  the  age  incidence  of  enlarged  spleens 

in  Central  Africa,  and  on  Chart  II  I.  are  shown  the  same 
cases  subdivided  into  two  widely  different  groups  ;  in 
the  one  district  Europeans  often  pass  their  first  year 
without  getting  malaria,  whilst  in  the  other  few  escape 
for  more  than  a  few  weeks.  The  earliest  age  incidence 
of  enlarged  spleen  and  the  earlier  period  at  which  it 
ceases  to  be  common  in  the  more  malarial  districts  are 
well  shown. 

CHART  III. 

Negroes  (Native  Africans),  in   a   most   Malarial  District 

in  Central  Africa.     Residence  required  for  Probahle 
Infection  with  Malakia,  under  Six  Weeks. 

Native   African,    in   less    Malarial    District.      Residence 

for  One  Year  does  not  render  Infection  Certain. 


YearL"      '  2      5          10        15        20       25       30 

M     90^ 

5      80- 

ca 

«<=  7°r 

■a  a6G" 
°&      J 
Z-o507 

0  <D 

it  40  _ 

cm 

v    20r 

o 

1  10V 

\ 

•J 

/ 

\*». 

r| 

\^ 

> 

The  weakness  of  the  spleen  test  is  that  a  considerable 
proportion  even  of  untreated  cases  of  malaria  do  not 
show  marked  splenic  enlargement,  and  it  is  probable 
that  68  per  cent,  of  enlarged  spleens  indicates  universal 
infection  as  much  as  90  per  cent,  harbouring  parasites 
would  do. 

Chart  IV.  indicates  the  proportion  of  persons  at 
different  ages  with  malarial  pigment  in  the  spleen  in  a 
moderately  malarial  country.  Two  years'  exposure  was 
required  for  probable  infection.  It  shows  malarial  infec- 
tion later,  and  less  complete  immunity. 


CHARTS 


469 


The  liability  to  malarial  infection  as  determined  by  the 
first  method,  the  length  of  residence  required  for  probable 
infection,  would  be  simply  charted  for  different  districts 
by  representing  for  each  place  in  the  vertical  lines  the 
number  of  months  requisite  for  the  infection  of  three- 
fourths  of  susceptible  newcomers,  or  the  percentage  of 
persons  who  would  be  infected  in  a  period  of  six  months 
or  a  year,  as  is  considered  to  be  most  convenient. 


CHART   IV. 

Negroes   (Native  Africans).     Compiled   from    Post-mortem 
Examinations  in  British  Guiana. 


Age  in      , 
Years 

2     5        10       15       20      25      30      35     40              50             60  Over  60 

a     80 

-C  in 

m  5  70  - 

0)    <D 

°co 

J 

-o50  _ 

P 

uS40- 

B  M30 

«      20- 
»      in  ' 

1 

Q_          10 

The  line  commences  at  one  month,  no  pigmentation  being  found  earlier. 
The  next  point  is  "  under  six  months." 

In  some  charts  the  distances  on  the  horizontal  line 
have  no  meaning,  and  it  is  simply  for  convenience  that 
the  horizontal  spacings  are  made  ;  a  series  of  vertical 
columns  packed  together  or  widely  and  irregularly 
separated  would  have  the  same  meaning  but  cause 
confusion. 

The  convenience  of  such  charts  is  that  various  points 
can  be  indicated  on  the  same  chart  and  compared. 

Graphic  Representations  of  Effect  of  Prophylactic  Measures. 
— The  success  that  has  attended  prophylactic  measures 
in  many  places  has  been  marked.  Some  of  the  observa- 
tions are  imperfect,  and  in  many  of  them  several  methods 
have    been    employed    and    have    been    associated    with 


470 


CHARTS 


improved  treatment  of  the  disease,  and  therefore  tin- 
actual  results  do  not  depend  entirely  on  a  reduction  of 
the  endemic  index. 

Two  illustrations  of  the  results  obtained  are  selected  for 
charting  and  criticism. 

One  is  that  recorded  by  Travers  and  Watson  for  the 
town    of    Klang    in    the   Malay   Peninsula.      The   figures 


CHART  v. 

Compiled  from  Reports  hy  Travers  and  Watson. — Journal  of  Tropical 

Medicine,  July  3,  1906. 

Indicates  number  of  cases  admitted  with  malaria  from  Klang. 

Ditto  from  surrounding  districts,  where  no  measures  had  been  taken. 


YEAR 

1901              1902            1903            1904           1905 

CASES 

350 

300 

250 

200 

1  50 

100 

50 
25 

1 

\ 

/ 

\ 

V,  m 

>^ 

• 

TZ  \\    N 

* 

• 

V 

• 

^ 

^m 

In  1901,  176  Government  officials  had  a  total  of  sick  leave  amounting  to 
1,026  days,  whilst  in  1904,  2S1  had  only  71  days'  sick  leave. 

reported  are  the  admissions  to  hospital  for  malaria 
verified  by  blood  examination,  and  the  deaths  attributed 
to  malaria,  in  both,  for  a  series  of  years. 

The  population  is  known  to  have  increased,  but  as  the 
amount  of  increase  is  not  known  it  is  taken  as  stationary, 
and  to  that  extent  the  results  appear  rather  less  striking 
than  they  really  are. 

The  measures  adopted  were  to  intercept  by  drains, 
running  across  the  base  of  a  hill  behind  the  town,  the 


CHARTS 


47 1 


water  that  would  otherwise  have  joined  the  town  subsoil 
water,  and  by  the  provision  of  numerous  deep  drains  to 
lower  the  level  of  the  subsoil  water  in  the  town.  Later 
hollows  were  filled  in.  The  effect  has  been  to  diminish 
the  number  of  certain  species  of  Anophcllnce;  species  such 
as  Myzomyia  rossi  are  still  very  abundant,  but  these  are 
not  efficient  carriers  of  malaria. 

CHART   VI. 

Deaths  from  malaria  in  the  drained  area. 

- Deaths  certified  as  from  other  diseases. 


YEAR 

1900              1901            1902             1903            1904            1905 

DEATHS 
120 

1  1  0 

100 

so 

80 
70 
GO 

50 

y 

i 

to  —  - 

h—  — i 

'*.  i 

\ 
\ 

V 

> 

During  the  same  period  in  the  surrounding  undrained  districts  the  death- 
rate  certified  as  from  fever  increased  from  173  in  1900,  to  351  in  1905,  and 
from  other  diseases  from  133,  to  271  in  1905. 

These  works  were  commenced  in  1901  and  continued 
since,  the  greater  part  being  supplied  early  in  1902,  and 
were  carried  out  by  the  Government  in  accordance  with 
recommendations  made  by  a  Committee  composed  of 
three  medical  men  and  three  engineers. 

The  main  results  are  indicated  in  the  charts  (V.  and 
VI.).  As  a  control  the  surrounding  district  where  no 
anti-malarial  measures  were  adopted  is  used.  It  will  be 
seen  that  whilst  in  the  towns  both  the  number  of  cases  of 
malaria  and  of  the  deaths  was  greatly  reduced,  though  the 


47-  KLANG    EXPERIMENTS 

population  had  increased,  in  the  surrounding  districts 
there  was  an  increase  in  both  these  items.  The  increase 
probably  was  due  to  the  increase  in  the  population. 

One  striking  feature  is  that  the  deaths  from  other 
causes  are  diminished  as  well  as  those  from  malaria,  indi- 
cating the  indirect  influence  of  malaria  on  the  prevalence 
and  severity  of  other  diseases.    This  is  shown  in  Chart  VI. 

The  numbers  are  considerable,  as  the  population  is 
over  4,000,  and  the  cases  of  malaria  were  334  in  1901, 
and  average  twenty-nine  from  1903  to  1904  with  a  popu- 
lation known  to  be  greater.  Applying  Poisson's  formula, 
we  find  that  dividing  the  population  4,000  into  two 
groups,  one  group,  334,  consisting  of  those  who  con- 
tracted malaria,  and  another  group,  3,666  consisting  of 
those  who  did  not  contract  malaria,  the  limits  of  error 
are  represented  by 

_|_   2    V  2  x  334  x  3,666  _         . 

—  ~~       4.000  x  4,000  x  400  pel  unit. 

=  i  '0123715  per  unit ; 

Or,  +  49^86  for  a  population  of  4,000. 

So  that  the  number  of  cases  of  malaria  per  annum  in 
this  population,  if  the  conditions  had  remained  con- 
stant, might  have  varied  between  334  4-  49  —  383,  and 
334  —  49  =  285.  As  will  be  seen,  the  reduction  to  29 
in  1903  is  manifestly  outside  the  limits  of  mathematical 
error,  and  indicates  a  marked  improvement  in  the  condi- 
tions by  which  malaria  was  spread  in  this  district. 

Any  error  therefore,  if  it  existed,  is  one  due  to  errors 
of  observations,  and  the  "  control "  is  a  sufficient  cheek 
on  this. 

Chart  VII.  indicates  the  incidence  of  disease  in  a 
series  of  Mission  stations  in  Central  Africa  for  a  period 
of  years.  From  1900  onward  various  anti-malarial 
measures  have  been  taken.  These  measures  varied  in 
different  stations,  and  were  carefully  planned  by  the 
medical  officer,  Dr.  Howard,  in  accordance  with  the 
local  conditions.  As  charted  the  effects  are  most  striking, 
as  the  proportion  invalided  or  who  died  during  the  li\'e 


CENTRAL    AFRICAN    EXPERIMENTS 


473 


years  when  these  measures  were  practised  was  less  than 
quarter  of  that  in  the  preceding  five  years.  The  actual 
numbers  are  very  small,  as  in  the  second  period  there 
were  only  an  average  of  24*4  workers  and  a  total  of  122 
in  the  five  years.  Of  these  7  died  and  8  were  invalided 
=  a  loss  of  15  ;  in  190 1  to  1905  there  were  182,  of  these 
2  died  and  4  were  invalided  =  a  loss  of  6. 


CHART   VII. 
Compiled  from  Report  by  Howard,  Published  London  School  of 

Tropical  Medicine,  February,  1907. 
Deaths  per  i,ooo.     Number  invalided  per  1,000. 


CALCULATED 

PER  1000 

120 

1  10 

100 

90 

80 

70 

60 

50 

4-0 

30 

20 

10 
0 

1887 

88 

89 

90 

91 

92 

93 

94- 

95 

96 

97   98 

99 

1900 

1 

2 

3 

4 

5 

r 
1 

1 

1 

l 

5 

I 
1 

l 
1 

1 

„„ 

-- 

_- 

«.,„ 

._- 

1 

l 

..... 

-_i 

1 

1 

i 

In  this  chart  the  figures  are  so  small  that  fluctuations  from  year  to  year 
are  considerable,  and  the  result  is  better  shown  by  averaging  for  periods  of 
five  years.  The  value  of  the  evidence  is  considerable,  but  the  small  figures 
available  reduce  this  value  to  a  great  extent. 

If  we  apply  Poisson's  formula  to  these  figures  we  find 
that  the  ratio  in  the  first  period  involved  a  probable 
error  of  '059552  per  unit  ;  in  a  population  of  182  in  a 
similar  period  the  number  of  deaths  might  have  been 
21,  or  a  minus  quantity.  The  reduction  to  two  there- 
fore falls  within  the  limits  of  mathematical  error. 

This  result,  therefore,  though   striking,  must  be  taken 


474  BLACKWATEK    FEVER    STATISTICS 

rather  as  an  illustration  than  as  a  proof  of  the  value  oi 
such  methods. 

Many  statistics  require  correction  before  charting.  Of 
these  corrections  some  are  obvious  and  easily  made. 
The  number  of  cases  is  useless  unless  the  total  number 
of  the  population  that  are  susceptible  to  the  disease  1- 
also  known.  In  dealing  with  inhabitants  of  different 
races  any  difference  in  the  susceptibility  has  to  be  noted 
and  allowed  for. 

Blackwater  fever  is  a  good  illustration  in  point,  and 
so  many  erroneous  statements  are  made  in  connection 
with  it  that  it  well  serves  as  an  illustration.  It  occurs 
in  Tropical  Africa,  in  India,  and  in  the  West  Indies, 
amongst  other  places.  All  races  are  probably  susceptible, 
though  this  is  doubted  by  some  as  regards  some  negro 
races  :  in  any  case  the  susceptibility  varies,  and  the  negro 
susceptibility  is  so  slight  that  not  one  in  many  thousands 
will  get  the  disease  under  conditions  where  some  8  per 
cent,  of  the  Europeans  are  attacked.  The  Indian  is 
certainly  susceptible,  but  only  about  one-fourth  as 
susceptible  as  the  European.  These  variations  in  racial 
susceptibility  require  much   further  study. 

We  must  know,  therefore,  both  the  number  of  Euro- 
peans and  the  number  of  cases  that  occur  amongst  them 
in  each  district  before  we  can  compare  the  prevalence 
of  the  disease  in  each  district.  Similarly  the  number 
of  cases  amongst  Indians  and  the  number  of  Indians 
must  be  known,  and  the  proportion  in  the  two  races 
must  be  kept  distinct. 

If  this  is  done  it  will  be  found  as  a  general  rule  that 
in  the  most  malarial  district  in  Africa  the  prevalence 
of  blackwater  fever  is  the  greatest,  though  the  actual 
number  of  cases  seen  may  be  no  more  than  in  a  more 
thickly  populated  but  less  malarial  district.  It  will  also 
be  found  that  it  is  only  in  Africa  that  <i  large  proportion 
of  susceptible  persons  are  attacked.  In  other  countries, 
where  perhaps  as  many  cases  may  be  seen,  the  number 
of  susceptible  persons  is  much  larger. 


BLACKWATER    FEVER   STATISTICS  475 

Another  correction,  an  important  one,  has  to  be  made. 
Unfortunately  the  amount  of  the  correction  is  dependent 
on  a  variable  factor — the  period  of  incubation  of  the 
disease.  More  cases  of  blackwater  fever  probably  occur 
in  England  than  in  any  one  district  in  Africa,  but 
these  cases  are  all  in  people  who  have  returned  from 
Africa  and  acquired  the  infection  there.  In  these  cases 
there  is  no  doubt  that  the  infection  should  be  attributed 
to  the  part  of  Africa  from  which  they  came.  Here  the 
matter  is  easy,  but  in  Africa  itself  it  is  so  often  found  that 
persons  develop  the  disease  who  have  been  travelling, 
that  it  is  a  matter  of  great  difficulty  to  attribute  the 
disease  to  the  correct  place  of  origin.  In  many  cases 
the  place  where  the  disease  develops  is  certainly  not  the 
place  where  it  was  acquired.  The  correction  to  be  applied 
here  is  essential,  but  can  only  be  an  approximate  and 
arbitrary  one  in  the  present  state  of  our  knowledge,  as 
the  period  of  "incubation"  is  unknown,  and  probably 
variable.  It  is  better  to  take  the  place  of  residence  a 
fortnight  before  the  attack  as  the  more  probable  place 
to  be  implicated  in  a  large  proportion  of  the  cases  than 
the  place  where  the  symptoms  of  the  disease  manifested 
themselves. 

Charting  is  often  useful  to  represent  the  secretion  or 
excretion  rates  either  of  definite  substances,  such  as 
urea,  or  the  volume  of  a  mixed  fluid,  such  as  urine. 
Here  times  are  represented  by  the  distance  measured 
horizontally,  and  amounts,  weights,  or  volumes  by  the 
height  measured  vertically. 

The  only  difficulty  is  that  however  it  may  be  secreted 
urine  as  well  as  other  fluids  are  only  passed  at  intervals, 
and  it  is  the  rate  at  which  urine  is  being  formed,  not 
that  at  which  it  is  being  passed,  that  is  of  importance. 
The  only  available  method  is  to  divide  the  number  ot 
ounces  of  urine  passed,  or,  if  necessary,  drawn  off  by 
catheter,  by  the  intervals  measured  in  hours  between 
the  successive  micturitions  ;  the  result  will  give  the 
average  rate  per  hour,  assuming  that  the  bladder  is  equally 
empty  after  each  micturition. 


47^  SECRETION    RATES 

Such  charts  arc  of  special  value  in  diseases  like 
blackwater  fever  and  yellow  fever,  in  which  there  is  a 
tendency  to  suppression  of  urine,  and  may  indicate  the 
periods  of  greatest  danger. 

The  geographical  and  topographical  distribution  of 
disease,  of  parasites  and  of  certain  insects  is  of  consider- 
able importance.  Maps  should  be  drawn  to  the  required 
scale  and  the  places  where  an  examination  shows  that 
the  condition  to  be  charted  is  present  marked  with  a 
plus,  +  ,  and  where  absent  negative,  — .  Places  where 
no  observations  have  been  made  should  be  clearly 
indicated,  for  if,  as  is  sometimes  done,  they  are  repre- 
sented as  negative,  most  misleading  conclusions  as  to  the 
distribution  of  the  disease  will  be  drawn. 

It  is  usual  to  represent  the  incidence  of  a  condition  by 
shading,  and  the  depth  of  the  shading  indicates  also  the 
prevalance  of  the  condition. 

In  determining  the  incidence  of  a  disease  in  a  town  or 
village  a  plan  must  be  drawn  up  and  the  houses  or  groups 
of  houses  infected  indicated  as  above. 

Extraneous  conditions,  such  as  wells,  streams  or  other 
sources  of  water,  must  be  shown,  and  when  dealing  with 
a  question  such  as  malaria,  known  to  be  carried  in  a 
certain  way,  other  conditions  favouring  the  prevalence 
of  such  carriers  as  Anophelince  must  also  be  indicated. 

These  maps  and  charts  enable  the  conditions  to  be 
quickly  understood,  and  are  therefore  of  considerable 
value  if  accurate  and  carefully  drawn  up. 

With  a  little  ingenuity  almost  anything  can  be  repre- 
sented in  a  graphic  manner,  or  charted.  The  value  of 
a  chart  is  the  ease  with  which  relations  are  shown  and 
from  which  conclusions  can  be  deduced.  They  show  no 
more  and  prove  no  more  than  the  figures  or  facts  they 
represent,  but  are  more  easily  followed  by  many.  If, 
therefore,  a  chart  does  not  represent  matters  more  clearly 
than  the  figures  the  chart  is  useless. 


477 


APPENDIX. 


Average  Weights  of  Organs  in  Ounces. 

Brain 
Europeans  (Quain)  ...     49/5 

Negroes    (British  Guiana)     42^04 
Indians     ...         ...  ...     4i'9 

Chinese     ...         ...         ...     47^3 

Variation  in  the  Weights  of  Lungs  with  the  Time  after  Death. 

No.  of  cases  in 


Heart 

Lung.i 

Spleen 

Liver 

Kidney 

I  I 

•  45 

.     6       . 

•  53 

..    II 

108  . 

■  237  . 

6-9  . 

•  477 

..      9"9 

87   . 

.  27-2  . 

.   18-4  . 

•  4^-4 

..     8-9 

9      ■ 

..  26"!   . 

..    14-8  . 

•  43  "4 

•  •     87 

Examination  made 

Average 

which  the  lungs 

21  ozs.  to 

31  ozs.  to 

41  ozs. 

ifter  death 

weighis 

weighed 

30  ozs. 

40  ozs. 

and  over 

20  ozs.  or  less 

With 

n    3 

hours 

..         18 

71 

21 

I 

— 

>> 

4 

)> 

•         23 

9 

25 

4 

I 

,, 

6 

!7 

..         27 

11 

32 

..    26     .. 

3 

) » 

12 

)) 

29M 

4 

37 

•  •     37       ■• 

6 

,, 

18 

)) 

..       34-8 

3 

47 

..    52     .. 

35 

Over 

18 

M 

44-1 

— 

3 

..       9       .. 

33 

Average  Weights  of  Brain  in  Negroes  and  Indians 
at  Different  Ages. 

Ages:  16  to  20  21  to  30  31  to  40  41  to  50  51  and  over 

Negroes...       44-3        ...  46  ...  45-4        ...  43  ...         41-5 

Indians  ...        40  ...  4i'2       ...  4i'3       ...         4C8       ...         40*5 

Proportion    of   the   Spleens    in    Indians   and   Negroes   Weighing 
15  ozs.  and  Over  in  British  Guiana. 

Ages  Indians  Negroes 

20  to  25  ...  30  per  cent.  ...  32  per  cent. 

26  to  45  ...  52         ,,  ...  16         ,, 

Over  45  ...  39          „  ...           15 

Preservation  and  Examination  of  Worms. 
Small  Nematodes  (up  to  the  size  of  and  including  Ankylostomes). 
Preservation. 

(1)  Shake   up  the   live    worms  in  a   1   per  cent,  salt  solution,  to 

remove  mucus,  &c. 

(2)  Kill  by  dropping  into  boiling  70  per  cent,  spirit  and  allow  to 

cool. 

(3)  Transfer  to  fresh  70  per  cent,  spirit  for  storage. 
Clearing  and  mounting  same. 

(1)  After  treating   with  stages   1    and  2  as  above,  transfer  to  a 


47$  EXAMINATION    OF    WORMS 

mixture  composed  of  70  per  cent,  spirit  95  parts  with  5  parts 
of  pure  glycerine. 

(2)  Evaporate  on  a  water  or  paraffin  bath  until  all  the  alcohol 

has  gone. 

(3)  .Mount  in  glycerine  jelly. 

(4)  Ring  with  gold  size. 
Examination  without  mounting. 

(1)  Take  from   the   70  per  cent,  spirit  and  place  in  methylated 

spirit. 

(2)  Transfer  to  colourless  coal  tar  creasote,  allow  to  clear,  and 

examine  in  that  fluid. 
After  the  examination    is  concluded    pass    through    methylated 
spirit  back  to  70  per  cent,  spirit  to  store. 

Large  Nematodes  are  treated  in  the  same  way  as  small  ones,  except 
that  they  cannot  be  cleared  and  mounted  by  the  glycerine 
method. 

Small  Trematodes. 
Preservation. 

(1)  Place  alive  in  a  test  tube  one-third  full  of  1  per  cent,  saline. 

and  shake  vigorously. 

(2)  Add  to  the  test  tube  rapidly  an  equal  quantity  of  saturated 

solution  of  sublimate. 

(3)  Shake  vigorously  for  three  minutes. 

(4)  Transfer  to  70  per  cent,  spirit  to  store. 
Examination  without  mounting.     As  for  small  nematodes. 

To  make  stained  and  mounted  specimens. 

(1)  Transfer  from  70  per  cent,  spirit  to  a  1  per  cent,  solution  of 

aium  to  which  is  added  a  little  hematoxylin,  until  the  whole 
is  of  a  light  claret  colour.  The  haemalum  solution  (page  52) 
diluted  with  distilled  water  gives  good  results,  and  so  does 
a  weak  solution  of  carmine,  but  in  that  case  longer  staining 
is  required.     Leave  in  this  one  to  four  days. 

(2)  Decolourise  slightly  with  \  per  cent,  acid  water. 

(3)  Wash  well. 

(4)  Dehydrate  with  spirit  and  oil  of  cloves. 

(5)  Pass  through  xylol  and  mount  in  balsam. 

Large  Trematodes. 
Preservation. 

(1)  Drop  alive  into  1  per  cent,  saline  and  shake  vigorously 

(2)  Add  formalin  (commercial)  to  this  to  make  about  a   10  per 

cent,  solution,  and  shake  vigorously  till  death  occurs. 
The  specimens  can  be  kept  in  10  per  cent,  formalin. 
Examination. 

This  is  best  done  by  embedding  and  cutting  sections. 


VARIOUS    STAINING    METHODS  479 

To  mount  specimens  whole. 

(i)  Press  between  two  slides  whilst  alive,  and  drop  into  70  per 

cent,  spirit. 
(2)  Stain  and  clear  as  in  small  specimens. 

Cestodes. 

Preservation. 

(1)  Shake  gently  in  1  per  cent,  saline. 

(2)  Add  formalin  to  this  to  make  a  10  per  cent,  solution  and 

shake  gently  till  they  die. 
The  specimens  can  then  be  stored  in  10  per  cent,  formalin. 
To  stain  and  mount  segments. 

(1)  Place  alive  in  weak  glycerine  faintly  coloured  with  carmine 

and  leave  till  stained,  or  dilute  haemalum  may  be  used. 

(2)  Press  between  two   slides  and  drop  into  methylated   spirit. 

They  should  remain  in  this  for  twenty-four  hours. 

(3)  Remove    the    pressure    and    place    the    segments    in    fresh 

methylated  spirit  for  an  hour  or  so. 

(4)  Clear  in  oil  of  cloves. 

(5)  Pass  through  xylol  and  mount  in  balsam. 
(Creasote  may  be  used  to  clear  instead  of  oil  of  cloves.) 

Various  Staining  Methods. 
A. — A   metJiod  for  Staining   Gregarines. 

(1)  Make  a  film  on  a  coverslip  and  keep  it  moist. 

(2)  Whilst  still  wet  drop  the  coverslip  face  downwards  on  to  the 
fixing  solution  consisting  of  two  parts  of  saturated  sublimate  solution 
and  one  part  of  absolute  alcohol.     Leave  it  to  fix  for  fifteen  minutes. 

(3)  Wash  in  70  per  cent  alcohol. 

(4)  Place  in  70  per  cent,  alcohol  to  which  a  few  drops  of  Gram's 
iodine  solution  have  been  added,  so  that  the  colour  of  the  mixture  is 
like  that  of  weak  tea.     Leave  in  this  ten  minutes. 

(5)  Place  in  70  per  cent,  alcohol  to  which  have  been  added  a  few 
drops  of  Delafield's  hematoxylin  and  leave  for  twenty-four  hours  or 
longer  as  required. 

(6)  Wash  in  70  per  cent,  alcohol. 

(7)  Wash  in  methylated  spirit. 

(8)  Remove  spirit  with  xylol  and  mount. 

B. — Levaditi's  Method  of  Staining  Spirochetes  in   Tissues. 

(1)  Pieces  of  the  tissue  1  mm.  thick  are  fixed  in  10  per  cent, 
formalin  for  twenty-four  hours. 

(2)  Wash  and  harden  in  96  per  cent,  alcohol  for  twenty-four  hours. 

(3)  Wash  in  distilled  water  till  tissue  sinks. 

(4)  Place  in  silver  nitrate  solution  (1*5  per  cent.)  for  three  to  five 
days  at  37°  C. 


480  VARIOUS    STAINING    .METHODS 

(5)  Wash  rapidly  and  place  at  room  temperature  for  twenty-four  to 
forty-eight  hours  in  : — 

Ac.  pyrogallic  ...  ...  ...  ...  2  to  4  gr. 

Formol  ...  ...  ...  ...  ...  5  c-c- 

Aq.  desiillata  ...      100  c.c. 

(6)  Wash  in  distilled  water  and  pass  through  absolute  alcohol  and 
xylol. 

(7)  Embed  in  paraffin  and  cut  sections. 

C. — Staini?ig  of  Amosbce  Cysts. 

(1)  Make  a  film  on  a  coverslip  and  do  not  let  it  dry. 

(2)  Whilst    still     wet    drop  it   face   downwards    on    to   the   fixing 
solution  : — 

Saturated  solution  of  sublimate        ...  ...     2  parts. 

Absolute  alcohol        ...  ...  ...  ...      I  part. 

Leave  it  to  fix  for  fifteen  minutes. 

(3)  Take  out  of  the  fixing  solution  and  place  in  40  per  cent,  alcohol 
for  ten  minutes. 

(4)  Place  in  70  per  cent,  alcohol  to  which  a  few  drops  of  Gram's 
iodine  solution  have  been  added.     Leave  in  this  ten  minutes. 

(5)  Place  in  methylated  spirit  for  ten  minutes. 

(6)  Place  in  70  per  cent,  alcohol  for  five  minutes. 

(7)  Place  in  40  per  cent,  alcohol  for  five  minutes. 

(8)  Place  in  water  for  an  indefinite  time. 

(9)  Place  in  iron-alum  solution  {ih  per  cent.)  for  two  to  three  hours. 

(10)  Rinse  lightly  in  water. 

(11)  Stain  for  two  to  three  hours  in  hematoxylin  solution  made  as 
follows  : — 

Hematoxylin  crystals  ...  ...  ...        1  grm. 

Absolute  alcohol        ...  ...  ...  ...      IO  c.c. 

Disiilled  water  ...  ...  ...  ...     90  ,, 

This  solution  should  be  kept  for  a  month  to  ripen.  Then  add 
another  100  c.c.  of  water. 

(12)  Wash  well  in  water. 

(13)  Differentiate  in  iron-alum  solution  ('25  per  cent.).  This  is  best 
done  in  a  watch  glass  with  just  enough  solution  to  cover  the 
underneath  surface  of  the  film.  The  process  can  be  watched  under 
the  microscope  with  a  |  objective. 

(14)  Dehydrate  in  spirit  of  gradually  increasing  strength  :  40,  70, 
90  per  cent.,  and  absolute. 

(15)  Pass  through  xylol  and  mount  in  Canada  balsam. 

D. — Antiformin  Method  for  Tubercle. 
Solution  A. — Liquor  sodas  chlorinatae  (Sodae  carbonas,  600  parts ; 
bleaching  powder,  400  parts  ;  water,  1,000  parts). 
Soluiio?i  B. — Caustic  soda,  15  per  cent,  solution. 

To  make  antiformin  take  equal  parts  of  each. 


SPINAL   PUNCTURE  481 

Method. 

(1)  Take  5  c.c.  of  sputum  and  5  c.c.  of  a  25  per  cent,  solution  of 

antiformin  and  mix  in  a  test  tube.     Allow  to  stand  for  twelve 
hours. 

(2)  Pour  off  the  supernatant  liquid  and  wash  with  saline.     Collect 

deposit  by  centrifuging. 

(3)  Wash  again  in  saline,  pour   off  excess  of  fluid  after  centri- 

fuging and  place  deposit  on  a  slide  previously  albuminised 
or  having  some  of  the  original  sputum  on  it. 

(4)  Allow  to  dry  in  the  air,  fix  with  heat  and  stain  with  Ziehl- 

Neelson  and  methylene-blue  as  for  tubercle. 

The  antiformin  destroys  all  bacteria  except  the  acid-fast  ones,  and 
the  advantage  of  the  method  is  that  it  allows  a  considerable  amount 
of  the  sputum  to  be  examined. 

Spinal  Puncture. 

Place  the  patient  on  his  right  side  with  the  knees  well  drawn  up. 
The  tips  of  the  fingers  of  the  left  hand  are  then  placed  upon  the  left 
iliac  crest,  when  the  thumb  will  indicate  the  site  of  puncture.  This  is 
at  a  point  between  the  fourth  and  fifth  lumbar  vertebras  and  lies  half 
an  inch  to  the  left  of  the  middle  of  a  line  joining  the  two  iliac  crests. 
Insert  a  stout  hypodermic  needle  for  one  or  two  inches  until  it  is  felt 
free  in  the  canal.  No  syringe  should  be  used  to  effect  suction,  but 
the  fluid  should  be  allowed  to  escape  naturally  through  the  needle. 

In  health  the  cerebro-spinal  fluid  is  of  a  pale  clear  colour  and 
contains  very  few  or  no  cells.  It  escapes  from  the  end  of  the  needle 
drop  by  drop,  whereas  in  cerebro-spinal  meningitis  and  cerebral 
tumours  it  usually  spurts  out. 

If  meningitis  is  present  the  fluid  is  turbid  and  contains  a  large 
number  of  leucocytes,  lymphocytes  predominating  if  it  is  tuberculous, 
whilst  the  polymorphonuclears  are  in  excess  in  other  forms  of 
meningitis. 

In  normal  cerebro-spinal  fluid  there  is  a  considerable  amount  of 
sugar,  whereas  in  meningitis  this  is  lost  or  reduced  to  a  mere  trace. 

Arrow  Poisons. 

(1)  Tetanus. — The  carcase  of  a  dead  animal  is  lightly  buried  under 
alternate  layers  of  earth  and  leaves.  After  some  time  has  elapsed  the 
carcase  is  uncovered  and  the  points  of  the  arrows  are  dipped  in  the 
decomposing  body.  This  poison  is  the  chief  one  in  use  among 
the  natives  of  the  South  Pacific  Islands. 

(2)  Antiaris  toxicaria,  or  Upas  tree. — The  inspissated  juice  is  used 
often  in  combination  with  strychnos,  chiefly  in  the  Malay  Archipelago. 
The  poison  is  harmless  if  taken  by  the  mouth,  but  when  injected 
causes  violent  intestinal  peristalsis  and  early  death. 

(3)  Curare. — A  decoction  of  Malonetia  nitida  used  chiefly  in  South 
America.  It  paralyses  the  peripheral  ends  of  the  motor  nerves  in  the 
voluntary  muscles. 

31 


4«s2  INSTRUMENTS   AND   REAGENTS 

(4)  Strophanthus. — Is  occasionally  used  by  natives  in  Africa. 

(5)  Strychnos  Ticuic. — A  decoction  of  the  bark  mixed  with  Antiaris 
toxicaiia  is  used  by  natives  in  the  Malay  States.  The  toxic  agent  is 
brucia. 

Instruments  and  Reagents. 

Microscope,  with  two  eye-pieces,  2  and  4  ;  three  objectives,  §  in., 
\  in.,  and  TV  in.  oil  immersion  lens ;  substage  condenser,  and  iris 
diaphragm  and  mechanical  stage,  micrometer  eye-piece  with  scales 
or  with  squares,  micromillimetre  scale,  camera  lucida. 

Watchmaker's  glass. 

Portable  microscope. 

Direct  vision  spectroscope. 

Slides,  No.  2  quality. 

Cover-glasses,  No.  1  quality,  to  be  packed  in  oil. 

Needles  in  handles.  Cork  felt.  Entomological  pins,  Xo.  20. 
Forceps.     Cornet's  forceps.     Mounted  platinum  wires. 

Test  tubes,  thick  and  best  quality.  Durham's  tubes.  Watch 
glasses.  Petri  dishes.  Photographic  trays,  half-  and  full-plate. 
Erlenmeyer's  flasks.  Funnels.  Glass  tubing.  Glass  rod.  Beakers. 
Burette,  50  c.c.  Evaporating  dishes  and  copper  dish  for  boiling 
slides.     Spirit  Bunsen.     Primus  kerosene  lamp. 

Glass  measures,  500  c.c,  100  c.c,  and  10  c.c. 

Scales.     Gramme  weights. 

Paraffin  oven.     Paraffin  moulding  dish  and  blocks. 

Microtome. 

Steam  steriliser.  Hot-air  steriliser  and  incubator.  Iron  enamelled 
jugs. 

Mounting  and  Embeddi7ig  Reagents. — Alcohol,  cotton  wool,  methy- 
lated spirits,  oil  of  cloves,  xylol,  Canada  balsam,  glycerine,  Farrant's 
solution,  glycerine  jelly,  ether,  chloroform,  celloidin,  paraffin  wax, 
Hollis's  glue,  or  shellac,  acetone,  aniline  oil,  creasote. 

Stains. — Haematoxylin  crystals,  hasmatein,methylene-blue(H6chst;s 
pure  medicinal),  thionin,  gentian  violet,  fuchsin,  carmine,  picrocar- 
mine,  toluidine  blue,  night  blue,  Bismarck  brown,  methyl  violet, 
acid  fuchsine,  eosine,  both  soluble  in  alcohol  and  soluble  in  water. 
Gram's  stain  made  up.  Leishman's  stain.  Eosin  azur  (Burroughs 
Wellcome's  '  tabloids').     N.B. — Grubler's  stains  are  the  best. 

Other  Reagents. — Acids  :  Hydrochloric,  nitric,  sulphuric,  picric, 
osmic,  tannic,  carbolic  (pure),  gallic,  sulphanilic,  pyrogallic  Agar- 
agar,  alum,  bleaching  powder,  ammonia,  alcohol,  methyl  alcohol  (pure 
for  analysis),  borax,  creasote,  iodine,  filter  paper,  filter  paper  (Chardin), 
formalin,  gelatine,  glucose,  sucrose,  mannitol,  lactose,  lithium  carbo- 
nate, lysol,  mercuric  chloride,  naphthaline,  peptone,  platinum  chloride, 
potassium  ferrocyanide,  potassium  iodide,  potassium  bichromate, 
phenolphthalein,  silver  nitrate,  sodium  carbonate,  sodium  citrate, 
sodium  hydrate,  sodium  sulphate,  sodium  taurocholate,  sodium  nitrite. 
Bovril. 


PLATE    IV. 


Stained  with   Hematoxylin  or   Eosine  and  Hematoxylin 

Figs. 

Normal  variations  in  red  blood  corpuscles. 

Nucleated  red  blood  corpuscles. 

Blood  plates. 

Abnormal  variation  in  size  and  colour. 

Abnormal  shapes,  poikilocytes. 

Basophilic  granules. 


6, 


Malignant  Tertian   Parasites  (Sub-Tertian)  Stained 
with  Carbol  Thionin. 


Young  form,  rings. 

Half-grown  parasite. 

Full-grown  parasite. 

Sporulating  parasite. 

Are  full  and  sporulating  parasites,  as  seen  in  sec- 
tions of  organs  shrunk  by  the  spirit  and  other 
processes. 

Development  of  the  gametes  of  malignant  tertian. 

Benign  tertian  parasites,  half-grown  and  sporu- 
lating. 

Quartan  parasites,  half-grown  and  sporulating. 

Sporozoa  of  cattle  and  horses. 


Figs 

10. 

II. 

12. 

14. 

13 

& 

15- 

16 

to 

19. 

20, 

21. 

22, 

23- 

24 

to 

27. 

Plate    IV. 


3. 


• 


10. 


n. 


•$£ 


12. 


•4 


13. 


14-. 


15. 


«Sw' 


16. 


■3$^ 


17. 


18. 


19. 


20. 


* 


21. 


23. 


2*. 


25 


26. 


27. 


A  Terz,    del 


Bale  &  DaiueJssorL.ltdLui 


PLATE    V. 

Stained  with  Leishman's  Stain. 

Figs. 

1.  Normal  red  corpuscle. 

2.  Blood  plates. 

3.  Lymphocyte. 

4.  Large  mononuclear  leucocyte. 

5.  Polymorphonuclear  leucocyte. 

6.  Eosinophile  leucocyte. 

7.  Mast  cell. 

8.  Transitional  form. 

9.  Abnormal    mononuclear    cell    found    in    certain 

diseases,  including  trypanosomiasis. 

Myelocytes  showing  various  types  of  granules. 

Halteridium. 

Small  drepanidium  in  red  corpuscle. 

Same  drepanidium  in  plasma. 

Large  drepanidium  in  various  stages. 

Degeneration  of  red  corpuscle  caused  by  drepa- 
nidium  (Schuffner's  dots). 


:o  to 

13- 

H. 

15- 

16. 

17- 

,   19. 

20. 

21. 

Plate  V. 


«' 


15 


19 


l»»t 


ftvV,  '    ,  '-"J 
10 


20 


s 


Ll 


11. 


* 


* 


18 


*. 


21 


A  Terzi    del 


Bale  &DaiuelssouLu  Ink 


r 


f 


PLATE   VI. 
Stained  with  Leishman's  Stain. 

Stages  of  benign  tertian  parasite. 

Gamete  benign  tertian. 

Characteristic  degeneration  of  red  corpuscles  con- 
taining benign  tertian  parasites  (Schiifmer's 
dots). 

Stages  of  quartan  parasite. 

Stages  of  malignant  tertian  (sub-tertian)  which 
are  seen  in  peripheral  blood. 

Male  gamete,  malignant  tertian  (sub-tertian). 

Female  gamete,  malignant  tertian  (sub-tertian). 

20.  Double   infection    with     malignant    tertian     (sub- 

tertian)  parasites  of  a  red  corpuscle  ;  Maurer's 
granules  in  red  corpuscle. 

21.  Spirillum  of  relapsing  fever   (stained  with  carbol 

fuchsin). 
22,  23.     Amceba  coli. 


Figs 

1   to 

5 

6. 

7,  8, 

9 

0  to 

*5 

16, 

*7' 

18 

19 

PJate   VI. 


•    ! 


"   »~  .  '    •'   ■ 


JO 


-}$> '. 


<°-- 


n 


I2 


13 


1-1- 


lS 


16 


17 


IS 


19 


20 


21. 


fti 


C 


JSk 


k 


% 


23 


#        \         * 


A  Terzi   del 


BtOe  &  Damelsscm  ItA  liUi 


489 


INDEX. 


Abdominal   and    thoracic    viscera,    removal    en  masse  in  post-mortem 

examinations     ...          ...          ...          •••          •■•  ■■■          •■•        2I 

Method  for «i  22 

Acari7ia  (mites  and  ticks)       ...          ...          ...          ...  29° 

Families  of,  characteristics         ...         ...          ...  ■••     297 

Acephalina       ...      109 

Acetone  and  paraffin  method  of  imbedding             ...          ...  ■••        33 

Adanal  plates 300-306 

s&deomyia        ...         ...         ...           . .          ...          •  •  •  •  •■     23° 

Characters  of        ...          ...          ...          ...          •■■  ...      231 

JEdes 2o8 

Character  of         ...          ...          ...          ...          ••■  23x 

ALdince : — 

Egg-laying  in  masses  or  rafts     ...          ...          ...          ■••  ...      261 

Larvre  of,  hairs  on  abdomen      ...          ...          ...           ••  ■••          ■•■     27I 

Respiratory  syphon  in         ...          ...          ...          •••  .271 

Acid  formation  in  bacteria     ...          ...          ...          ..  •■■     4°5 

Africa  : — 

Transmission  of  Piroplasvia  bigeminum  in      ...           ..  ...          •••      I03 

See  also  Monkeys,  African. 

Africa,  South,  carrier  of  Piroplasma  cants  in          ...          ...  ...                  i°3 

African  relapsing  fever  caused  by  ticks        ...          ...          •  ••  •••          ■••     3°3 

Agar:— 

Neutral  red           •••     400 

Nutrient,  see  Nutrient  agar. 

Plating  with         : ■■■     387 

Agglutinins       ...          ...          ...          ...          ...          ..             ••  ■••          ■••      '5° 

Agrionida : — 

Breeding  places  of           ...          ...          ...          ...          •••  '5" 

Larvae  of  ...         ...          ...         ...         ...          ...         ...  •••     '5° 

Air  and  mixing  chambers,  Wright's  tubes  with,  estimation  of  isotonic 

strength  of  blood  serum  by     ...          ...          ...          ...  •■•      MS 

Albumin  and  glycerine  method  of  fixation  ...          ...          ...  ■••       39 

Alcohol  :  — 

Absolute,  as  solvent  in  staining            ...         ...          ...  66 

Fixation  by  . . .          ...          ...          ...          . .           ...  •        51 

And  glycerine,  examination  of  Nematodes  in  .           ...  i 34«  '35 

And  paraffin  method  of  imbedding       ...          ...  3° 

Fixation  and  hardening  of  tissues  in     ..           ...          ...  ...          ■••        27 

Formol,  for  rapid  fixation  of  tissues     ...          ...           ..  28 

Preservation  of  Nematodes  in   ..           ...          ...  '34 


490 


INDEX 


I  A'.h 

Amllyomma  : — 

<  icnus  of  /xod/nu-            ...          ...          ..  ...  ...  ...  ...  306 

Hdnicuiii,  causing  heart-water  in  sheep  and  goals  ...  304 

America,  transmission  of  I'iroplasma  bigetninum  in  ...  ...  ...  103 

Ammonia,  albuminoid,  in  water,  test  for   ...  ...  ...  ...  431 

Free,  determination  of,  in  water           ...  ...  ...  ,..  430 

Estimation  of,  by  Wanklyn's  process  ...  ...  ...  430 

Amccbacoli       ...         ...         ...         ...         ...  ...  ...  ..  ...  346 

Found  in  stools    ...           364 

In  dysentery         365 

Life-history  of      ...          ...          ...          ...  ...  ...  ...  ...  365 

Study  of  stained  specimens  of  ...          ...  ...  ...  365 

Amoeba;  cysts,  method  of  staining    ...                       ..  ...  ...  480 

Amphistomum  or  Paramphistomuvt             ...  ...  ...  ...  ...  356 

Amyloid  degeneration             ...          ...          ...  ...  ...  ...  ...  319 

In  leprosy             ...          ...          ...         ...  ...  ..  ..  319 

Staining  for          ...         ...          ...          ...  ...  ...  ...  ...  319 

Anjemia  :  — 

Blood  counts  in    ...          ...         ...          ...  ...  ...  ...  ...  442 

Increase  of  eosinophiles  in          ...  ...  ...  ...  ...  58 

Nucleated  red  corpuscles  in  blood  in    ...  ...  ...  ...  53 

Pernicious,  blood  count  in         ...         ...  ...  ..  ..  ...  4^2 

Changes  of  red  blood  corpuscles  in  ..  ...  ...  ...  61 

Yellow  deposit  in     ...          ...         ...          ...  ...  ...  313,314 

Tropical,  enumeration  of  red  corpuscles  in  ...  ...  ...  ...  442 

Ankylostomiasis,  see  Ankylostomiasis. 

Aneurisms,  verminous,  in  horses       ...         ...  ...  ...  ...  ...  124 

Anguillula  intestinalis  (see  also  Slrongyloides)  ...  ..  ...  ...  361 

Animals,  rare,  grouping  and  classification  by  means  of  precipitin  test...  151 

Anisocheleomyia           ...          ...          ...          ...  ...  ...  ...  ...  231 

Ankyloslomiun  duodenale        ...         ...         ...  ...  ...  ...  32S,  329 

Characteristics  of  eggs  of            ...          ..  ...  ...  ...  341,  342 

Ankylostome,  eggs  of...          ...         ...          ...  ...  ...  ...  ...  360 

Ankylostomiasis  :  — 

Blood  count  in     ...         ...         ...         ...  ...  ...  ...  ...  443 

Fatty  degeneration  in     ...         ...          ...  ...  ...  ...  ...  319 

Leucocyte  variation  in    ..           ...          ...  ...  ...  ...  ...  59 

Yellow  deposit  in              ..         ...         ...  ...  ...  ...  3 1 3-3 1 5 

Ankylostomum  duodenale,  description  of  ...  ...  ...  ...  358 

Anopheles          ...         ...         ...         ...         ...  ..  ...  ...  ...  130 

Egg  of       263 

Larvce  of,  breeding  place  for  ("  Anopheles  "  pool)  ...  ...  ...  277 

Salivary  glands    ...          ...          ...          ...  ...  ...  ...  ...  250 

A nopheles  maculipennis          ...          ...          ..  ...  ...  ...  ...  278 

Anophelina       ...          ...          ...          ...          ...  ...  ...  ...  ...  253 

Feeding  time        ...          ...          ...         ...  ..  ...  ...  ...  159 

Anophelina       ...         ...         ...         ...         ...  ...  ...  ...  ...  234 

Eggs  of     ...          ...         ...         ...          ...  ...  ...  ...  ...  262 

method  of  laying     ...          ...         ...  ...  ...  ...  ...  262 


INDEX  491 

PAGE 
Anophelincz — continued. 

In  malaria             ...          ...          ...          ...  ...  ...  ...  ...  460 

Larva;  of,  a  syphonate    ...          ...          ...  ...  ...  ...  269 

Hairs  on  abdomen  in          ...         ...  ...  ...  ...  271 

Anopleura        ...         ...        ...         ...         ...  ...  ...  ...  291 

Characters  of        ...          ...          ...          ...  ...  ...  •••  •■•  '57 

Ant,  characters  of        ...          ...          ...          ...  ...  ...  ...  ...  157 

Anthomyia,  in  man     ...          ...          ...          ...  ...  ...  ...  ...  160 

Ant  homy  ides,  characters  of    ...          ...          ...  ...  ...  ...  ...  195 

Antiaris  toxicaria  (upas  tree),  arrow  poison  made  from    ...  ...  ...  481 

Antiformin  method  of  staining  for  tubercle...  ...  ...  ...  ...  480 

Advantages  of      ...          ...          ...          ...  ...  ...  ...  ...  481 

Antitoxins        ...         ...         ...         ...          ...  ...  ...  ...  15° 

Aponomma,  genus  of  Ixodinte           ...         ...  ...  ...  ...  ...  306 

Ap/era,  characters  of  ...          ...          ...          ...  ...  ...  ...  .  156 

Arachnida          ...          ...          ...          ...          ...  ...  ...  ...  ...  155 

Arachnoidea     ...          ...          ...          ...          ...  ...  ...  ...  ...  296 

Cephalothorax  of...          ...          ...         ..  ...  ...  ...  ...  296 

Araneidte  (Spiders)      ...          ...          ...          ...  ...  ...  ...  ...  296 

Argas,  genus  of  Argasinae      ...          ...          ...  ...  ...  ...  ...  3°4 

Argasinse           ...         ...          ...          ...          ...  ...  ...  ...  297-304 

Arista,  description  of ...          ...          ...          ...  ...  ...  ...  ...  161 

Arrow  poisons...          ...         ...         ...          ...  ...  ...  ...  ...  481 

Arsenic  in  water,  test  for       ...         ...         ...  ...  ...  ...  ...  429 

Antennae  of  fleas          ...          ...         ...         ...  ...  ...  ...  ...  285 

Arthropoda       ...          ...          ...          ...          ...  ...  ...  ...  154 

Groups  of...         ...         ...          ...         ...  ...  ...  ...  ...  155 

Ascaris  duodenale,  eggs  of     ...          ...          ...  ...  ...  ...  ...  341 

Ascaris  himbricoides,  eggs  of            ...          ...  ...  ...  ...  341,  342 

Mystax      357 

Aschiza,  characters  of             ...          ...          ...  ...  ...  ...  ...  167 

AsilidcB,  characters  of             ...         ...         ...  ...  ...  ...  ...  161 

Aspergillus        ...          ...          ...          ...          ...  ...  ...  ...  ...  420 

Causing  pneumomycosis              ...          ...  ...  ...  ...  ...  420 

Fumigattis            ...          ...          ...          ...  ...  ..  ...  ...  420 

Causing  pellagra       ...          ...          ...  ...  ...  ...  ...  420 

Assam,  hremogregarine  in  leucocytes  of  dogs  in  ...  ...  ...  ...  105 

Atoxyl,  Rhodesian  trypanosomiasis  resistant  to  ...  ...  ...  ...  117 

Alylotus,  character  of...         ...         ...          ...  ...  ...  ...  ...  175 

Auchmeromyia,  character  of...          ...          ...  ...  ...  ...  ...  194 

Auchmeromyia  luteola.;.          ...          ...          ...  ...  ...  ...  ...  194 

Bacilli,  intestinal  tubes  of  mosquitoes  contain  ...  ...  ...  ..  259 

Bacillus  coli  communis  ...         ...         ...  ...       370,406,407,413,417 

Diphtheria:            ...          ...          ...          ...  . .  ...  ...  ...  406 

Pestis,  fleas  carriers  of    ...          ...          ...  ...  ...  ...  ...  283 

Rats  host  of...          ...         ...          ...  ...  ...  ..  ...  283 

Typhosus 406,407,413,417 


492  [NDEX 

PAGE 

Bacteria : — 

Formation  of  acid  in        ...          ...         ...          ...  ...  405 

Of  gas  in      ...          ...         ...         ...          ...  ...  404 

Imbibed  by  larva,  subsequent  distribution  by  imago.  260 

In  urine    ...          ...          ...         ..           ...         ...  ...  375 

(Pathogenic),  examination  of  blood  for            ...  ...  139 

See  also  Organisms. 

Bacteriology      ...          ...          ...          ...          ...          ...  ...  378 

Apparatus  required  in  Tropics  for  simple  study  of     ...  378 

Description  of  organisms            ...         ...         ...  ...  388 

Methods  of  observing  important  points           ...  ...  388 

Method  of  work  ...          ..           ...         ...          ...  ...  ...                  378 

Preparation  of  films        ...          ...          ...          ....  389 

Separation  of  organisms...          ...          ...          ...  ...  385 

Balantidium  coli,  characteristics  of ...          ...          ...  ...  ...          ...     369 

Balfour,  description  of  mammalian  heemogregarines  ...  ...                  106 

Bat,  parasite  of            ...          ...          ...          ...          ...  ...  199 

Bat-ticks,  characters  of           ...          ...          ...          ...  ...  ...      167 

Bed-bugs,  characters  of          ...           .           ...          ...  ...  ...                   157 

Bee  :  — 

Characters  of       ...          ...          ..           ...         ...  ...  157 

Parasite  of            ...         ...         ...         ...          ..  .  .  ...      199 

Bee-louse,  characters  of         ...          ...          ...          ...  ...  ...      167 

Beetle,  characters  of  ...          ...          ...         ...         ...  ...  ...          1 55,  157 

Bentley,  discovery  of  Lencocytozoon  (Hcemogregariiia)  amis,  by...  ...      105 

Bentley-Taylor  method  of  mounting  mosquitoes  ...  ...  ...         235,236 

Benzol  and  chloroform  used  for  estimating  specific  gravity  of  blood      ...      142 
Beriberi :  — 

Changes  in  urine  in         ...          ...          ...          ...  ..  ...      376 

Leucocyte  variation  in    ...          ...         ...         ...  ..  ...       59 

Bichromate  solutions,  fixation  of  tissues  by            ...  ...  ...       23 

Bile  acids,  in  feces,  test  for,  Pettenkofer's  reaction  ...     338 
Bile  pigments  in  fasces: — 

Detection  by  Schmidt's  reaction           ...          ...  ...     333 

Gmelin's  reaction    ...          ...          ...          ...  ...  ...     333 

Iluppert's  test         ...         ...          ...          ...  ...  ...     333 

Bilharzia  infection,  increase  of  eosinophiles  in        ...  ...  ...          ...        58 

Bilirubin           333 

Biliverdin          ...         ...         ...          ...         ...          ...  -...  ...     333 

Birds  :— 

Blood-plasma  of  trypanosomes  in         ...         ...  ...  ...                  no 

Filarial  in  ...          ...          ...          ...          ...          ...  ...  ...          ...      259 

Halteridium  in      ..           ...          ...          ...          ...  ...  100.  101 

Position  of  filarial  in        ...          ...          ...          ...  ...  ...          ...      131 

Proteosoma  in      ...         ...         ...         ...          ...  ...  ...      100 

Blackwater  fever          ...          ...          ...          ...          ...  ...  ...     372 

Decrease  of  tonicity  of  blood  in...          ...          ...  ...  ...          ...      145 

Geographical  distribution  of      ..           ..           ...  ...  ...          ...      373 

Methaemoglobin  in           ...         ...         ...         ...  ...  ...         ...     143 


INDEX  493 

PAGE 
Blackwater  fever — continued. 

Mild  cases  overlooked     ...          ...          ...          ...          ...         ...  •••     374 

Period  of  incubation  of  ...          ...          ..           ...          ...          ...  ...     475 

Prevalence  of,  method  of  ascertaining...          ...          ...         ...  ...     474 

Statistics  dealing  with 474,475 

Susceptibility  of  various  races  to           ...          ...          ...         ...  ■•      474 

Value  of  charts  in            ...         ...          ...         ...         ...         ...  ■•■     47° 

Blastomycetes  (yeasts)             ...          ...          ..           ...          ...  ■•       421 

See  also  Yeasts. 

Blastophores     ...          ...          ..           ...          ...          ...          ...          ...  ••       254 

Blepharoceridce,  characters  of            ...          ...          ...          ...          ...  •••      169 

Blood  :— 

Amount  of,  in  disease     ...         ...          ...         ...          ...          ..  444 

Animal  parasites  found  in          ...          ...          ...         ...          ...  71 

Bodies  in,  mistaken  for  parasites          ...          ...         ...          ...  •■■       46 

Chemical  reaction,  determination  of 1-12,143 

Coagulation  of,  method  of  preventing  ...          ...          ...          ...  ...      141 

Coagulation  time  of        ...         ...         ...         ...          ...          •■•  •••     142 

Composition  of    ...          ...          ...          ...          ...          ...          •■■  •••       4° 

Containing  malaria  parasites,  permanent  preparations          ...  ...       94 

Dilution  of  150 

For  counting  leucocytes  or  red  corpuscles           ...          ...  ...     150 

Diseases  of,  variations  of  leucocytes  in            ...          ...          ...  57,  59 

Drawn  from  vein  by  hypodermic  syringe,  cultivation  of  organisms 

from 139,  140 

Examination  of    ...          ...          ...          ...          ...          ...          ..  ...       40 

For  crescent  bodies             ...          ...         ...          ...          ...  252,253 

For  malaria  parasites,  mistakes  in             ...          ...          ...  ...        96 

For  pathogenic  bacteria      ...          ...          ...          ...          ...  ...      139 

For  protozoa            ...         ...          ...          ...          ...          ...  ...       71 

Methods  72 

Hindered  by  coagulation     ...         ...         ...          ...          ...  •■•     141 

Spectroscopic           ...         ...         ...          ...         ...          ...  ...      143 

For  trypanosomes    ..           ...         ...         ...          ...         ...  ..       114 

For  injection  into  animals,  method  of  obtaining         ...          ...  141,   142 

Fresh,  methods  of  examination  of        ...          ,..          ...          ...  4J>44 

Staining  of   ...         ...         ...          ...          ...         ...          ...  ...       47 

Haemoglobin,  colouring  matter  of         ...           ..          ...          ...  ...      144 

(Human),  non-protozoal  parasites  found  in      ...          ...  ...      124 

Developmental  changes  in  filarial  embryos  not  effected  in  ...      129 
Immunization  in  one  species  against  that  in  another  closely  allied,  150,  151 
In  one  species  against  that  in  another  closely  allied,  new  method 

of  grouping  rare  animals             ...          ...          ...          ...  ...      15  [ 

In  faeces    ...         ...         ...          ...          ...          ...          ...          ...  33i»  332 

Weber's  test             ...          ...          ...         ...          ...          ...'  ...     332 

In  urine,  result  of  parasitic  invasions   ...          ...          ...          ...  ...      371 

Leishman-Donovan  bodies  in    ...          ...         ...          ...         ...  120 

Laking  of 143,  152 

definition       ...          ...          ...          ...          ...          ...          ...  ...      146 


494 


INDEX 


PAGE 
Blood — continued. 

Normal,  proportions  of  leucocytes  in   ...          ...  ...  56 

Of  animals,  parasites  in...          ...          ...          ...  ...  ...  100 

Of  lower  animals,  Nematodes  in           ...          ...  ...  ...  ...  124 

Parasites  in,  occurrence  of         ...          ...          ...  ..  ...  40 

Staining  of    ...          ...          ..           ...          ...  ...  ...  ...  62 

Ross's  method  of  measuring       ...          ...          ...  ...  ...  ...  445 

Specific  gravity  of  fluids  used  to  estimate        ...  ..  ...  142 

Specific  gravity  of           ...         ...         ...         ...  ...  ..  ...  142 

Blood-cells,  auto-agglutination  in  trypanosomiasis  ...  ...  ...  115 

Blood-changes  in  various  diseases     ...         ...         ...  ...  ...  57,  59 

Blood-corpuscles,  containing  malarial  parasite,  how  affected  ...  88,  89,  90 

Polychromatic,  staining  of         ...          ...          ...  ...  ...  ...  53 

Red,  changes  in  chlorosis           ...          ...          ...  ...  ...  ...  61 

Changes  in  leucocythremia             ...         ...  ...  ...  ...  62 

Malaria              61 

Pernicious  anaemia      ...         ...         ...  ...  ...  ...  61 

Crenation  of...         ...         ...          ...          ...  ...  ...  ...  45 

Diluted  blood  used  for  counting    ...          ...  ...  ...  ...  150 

Effect  of  heat  upon...         ...         ...          ...  ...  ...  ...  51 

Examination  of ...          ...          ...  ..  ...  ...  41 

Hemoglobin  in,  solution    ...          ...          ...  ...  144,  145 

Nucleated,  when  found       ...          ...          ...  ...  ...  ...  53 

Removal  of  haemoglobin  from        ...          ...  ...  ...  ...  144 

Staining  of    ...          ...          ...          ...          ...  ...  ...  ...  53 

Tonicity  of    ...          ...          ...          ...          ...  ...  ...  ...  144 

Decrease  in  blackwater  fever             ...  ...  ...  ...  145 

Importance  of  ...          ...          ...          ...  ...  ...  ...  145 

Index  to             ...          ...          ...          ...  ...  ...  ...  144 

Variation  in      ...          ...          ....      ...  ...  ...  ...  145 

Estimation            ...         ...          ...  ...  ...  ...  145 

Variations  in  shape  and  size  of      ...          ...  ...  ...  45.40 

Separation  for  examination         ...          ...          ...  ...  ...  ...  142 

Shadow,  staining  of        ...          ...         ...         ...  ...  ...  ...  53 

Vacuolated            ...          ...          ...          ...          ...  ...  ...  ...  45 

Blood-counts,  charts  used  in ...          ...          ...          ...  ...  ...  ...  466 

In  ancemia            ...         ...         ...         ...         ...  ...  ...  ...  442 

Ankylostomiasis       ...         ...          ...          ...  ...  ...  ...  443 

Malaria          442 

Leucocyte...          ...          ...          ...          ...          ...  ...  ...  443 

Method  of  enumerating ...          ...                      ...  ...  437,446 

Parasites  in   ..,          ...          ..           ..           ...  ...  ...  ...  444 

Methods  used  for             448,449 

Blood-films,  contamination  with  skin-organisms  during  preparation  ...  139 

Dried          72 

Examinations  for  filaria.'      ...          ...          ...  ...  ••■  ••  126 

Preparation   ...          ...          ...          ...          ...  ...  ...  ...  48 

Examination  under  microscope             ...          ...  ...  ■  •■  12 

Fixation  of,  methods       ...          ..           ...          ...  ...  ...  ...  51 


INDEX  495 

PAGE 

Blood  films — continued. 

Fixed,  treatment  of         ...  ..  ...         ...  ...  66,  67 

•  Fresh,  staining  of             ...           ..            ...          ...           ...                        ...  47 

Methods  of  making  ...  ...  ...  ...  ...  ...  41,  44 

Micro-fil.irice  in    ...          ...          ...          ...          ...          ...                       ...  125 

Preliminary  fixation  required  before  staining  ...                       ...          ...  72 

Not  required  before  staining           ...          ...          ...          ...          ...  75 

Preparation  of      ...          ...           ...           ...           ...           ...           ...           ...  389 

Fixation  and  staining  effected  together    ...          ...           ..          ...  74 

Staining  of  ...  ...  ...  ...  ...  ...  ...51-54,71,72 

Thickness  of,  variation  in           ...          ...          ...          ...          ...          ...  48 

Transparency  of  ...          ...          ...          ...          .  .          ...          ...          ...  144 

Blood-plasma  : — 

Hremogregarines  in          ..           ...          ...          ...          ...          ...          ...  110 

Human,  not  red  corpuscles,  invaded  by  spirocha;ta,  in  relapsing  fever  1 19 

Nature  of  ...          ...          ...          ...          ...          ...                       ...          ...  141 

Parasites  found  in            ...           ..         ...          ...          ...          ...          ...  no 

Piroplasma  in       ...          ...          ...          ...          ...          ...                       ...  110 

Trypanosomes  in...          ...          ...          ...          ...          ..,          ...          ...  110 

Blood-platelets  :  — 

Characteristics  of...          ...          ...          ...          ...          ...          ...          ...  47 

Staining  of  ...  ...  ...  ...         ....  ...  ...  53,  69 

Blood-serum  : — 

Amount   of  opsonin    present    in,    for   given  organism,    method    of 

estimation          ...          ...          ...          ...          ...          ...          ...          ...  152 

As  culture-medium          ...          ...          ...          ...         ...          .  .          ...  152 

Combination  of  broth-culture  with        ...          ...          ...          ...          ...  149 

Haemoglobin  in,  determination  of  presence  or  absence          ...          ...  143 

Diluted,  haemoglobin  in,  medium  for  cultivation  of  trypanosomes  ...  152 

Dilution  of             ...          ...          ...          ...          ...          ...          ...          ...  14S 

Method          148 

Repeated       ...          ...          ...          ...          ...           ..                        ..  149 

Dissolution  of  haemoglobin  in  (laking  of  blood)          ...          ...          ...  146 

Examination  of    ...          ...          ...          ...          ...          ...          ...          ...  143 

Hypertonic            ...          ...          ...          ...          ...          ...          ...          ...  146 

Method  of  obtaining        ...          ...          ...          ...          ...          ...          ...  147 

Power  of  effecting  destruction  of  pathogenic  organisms  by  leucocytes  152 

Substances  formed  as  result  of  infection  by  micro-organisms            ...  150 
Blood-vessels,  location  of  Microfilaria  bancrofti  in,  during  absence  from 

peripheral  blood           ...          ...          ...          ..           ...          ...          ...  129 

Malaria  parasites  found  in          ...          ...          ...          ...          ...          ...  87 

Blueing  films    ...         ...          ...          ...         ...          ...          ...          ...          ...  53 

Body-louse        ...          ...          ...          ...          ...          ...          ...          ...          ...  252 

Body,  organs  of,  variation  in  weight,  in  health  and  disease,  in  Tropics, 

from  European  standard         ...          ...          ...          ...          ...          ...  22 

Boiling  method  of  rapid  fixation        ...          ...          ...          ...          ...          ...  29 

Bolbodeomyia    ...          ...          ..           ...          ...          ...          ...          ...          ...  232 

Book-scorpions            ...          ...          ...          ...          ...          ...          ...          ...  296 

Roophilus,  genus  of  RhipicephaLc     ...         ...         ...         ...         ...         ...  306 


4<>" 


INDEX 


Borax  methylene  blue,  staining  with 
Bot-flies,  characters  of 
Bothriocephalns 

Mansoni   ... 
Bottles,  preservation  of  tissues  in 
Bouche,  characters  of  Homalomyia  ... 
Box  for  carriage  of  mosquitoes 
Brachycera  :  — 

Characters  of 

Classification  of    ... 

Description  of  families  ol 

Anomala,  characters  of  ... 

Vera,  characters  of 
Braddon's  method  of  blood  examination 
Brain  :  — 

Malaria  parasites  most  commonly  found  post  mortem 

Weight  of,  in  Europeans,  age  at  which  maximum  is  attained 
In  Negro,  age  at  which  maximum  is  attained 
Brauer,  classification  of  diptera 
Braula  ... 
Braulidoe 

Breeding-places  of  mosquito-larvce,  permanent 
Temporary 
Artificial 
Breeze-fly,  characters  of 
Brimp,  characters  of    ... 
Broth,  see  Nutrient  broth. 
Broth-cultures,  combination  wiih  blood-serum 

Points  to  be  observed  in 
Brulots  ... 

Brushes  on  head  of  larvre  of  mosquitoes 
Buffalo  :  — 

Trypanosoma  brucei  harboured  by 
non-pathogenic  to 
Buffalo-gnats    ... 

Characters  of 
Bunsen  burner,  automatic,  for  methylated  spirit     ... 
Butterfly,  characters  of 


...  72 

...  167 

...  349 

-  345 

...  27 

95.  196,  197 
...     280 

...  167 
...  166 
...  174 
...  167 
...  167 
42,  47 

Si,  84,  85 

22 

22 

166 

197 

199 

275 
275,  277 
277,  278 

174 
174 

149 
401 
172 
269 

112 
112 
172 
167 
3 
'57 


Cage  for  carriage  of  mosquitoes 
Calliphora,  in  myiasis 
"  Calyptrate,"  definition  of   ... 
Cambridge  rocking  microtome 
Camera  lucida  in  microscope  work  ... 
Canada  balsam  for  mounting  mosquitoes 
Capybara    (large    water-vole)    probable    natural    host    of 
equimim    ... 


Trypanosoma 


28 1 

160 

165 

37 

13 

234 


INDEX  497 

Carbol  fuchsin  : —  pack 

For  staining  trypanosomes         ...  ...  ...  ...  ...  114,115 

Staining  of  micro-filaria:  with    ...  ...  ...  ,..  ...  ...  127 

Carbol  thionin,  staining  with...          ...  ...  ...  ...  ...  ...  73 

Carriers  of  disease  : — 

Crustacea  as          ...          ...          ...  ...  ...  ...  ...  ...  309 

Ticksas     •••         3°3>  3°4 

Cathcart's  freezing  microtome           ...  ...  ...  ...  ...  ..  34 

Cattle  :— 

Often  recover  from  surra            ...  ...  ...  ...  ...  ...  112 

Texas  fever  of  (see  Texas  fever). 

T) ypanosovia  nanum,  parasitic  in  ...  ...  ...  ...  ...  113 

Cecidomyidte,  character  of     ...          ...  ...  ...  ...  ...  ...  168 

Cellia,  characters  of    ...          ...          ...  ...  ...  ...  ...  ...  225 

Cellia  albimana           ...          ...          ...  ...  ...  ...  ...  ...  225 

Cellia  a?-gyrotarsis       ...          ...          ...  ...  ...  ...  ...  ...  225 

Carrier  of  Filaria  noctiima        ...  ...  ...  ...  ...  ...  256 

Cellia  kochi      ...          ...          ...          ...  ...  ...  ...  ...  ...  225 

Cellia  pharcensis          ...          ...          ...  ...  ...  ...  ...  ...  225 

Celloidin  :  — 

For  demonstration  of  mosquitoes  ...  ...  ...  ...  ...  247 

Imbedding  method          ...          ...  ...  ...  ...  ...  ...  33 

Sections,  cutting  of         ...         ...  ...  ...  ...  ...  ...  38 

Cells,  abnormal,  resembling  leucocytes,  in  disease...  ...  ...  ...  60 

Degenerative  processes  in          ...  ...  ...  ...  ...  317-320 

Hyaline,  of  leucocytes    ...         ...  ...  ..,  ...  ...  ...  55 

Centipede,  characters  of         ...          ...  ...  ...  ...  ...  ...  155 

Cephalina          ...          ...          ...          ...  ...  ...  ...  ...  ...  108 

Cephalothorax  of  Arachnoidea          ...  ...  ...  ...  ...  ...  296 

Ceratophyllus  ...         ...         ...         ...  ...  ...  ..  ...  288,289 

Fasciatus  ...          ...          ...          ...  ...  ...  ...  ...  ...  290 

Ceratopogoji       ...          ...          ...          ...  ...  ...  ...  ...  ...  170 

Cercomonas  hominis,  description  of. ..  ...  ...  ...  ...  ...  367 

Cerebro-spinal  fluid,  condition  in  meningitis  ...  ...  ...  ...  48r 

In  health  and  disease      ...         ...  ...  ...  ...  ...  ...  481 

Cestoda...         ...         ...         ...         ...  ...  ...  ...  ...  ...  345 

Cestodes,  human          ...         ...         ...  ...  ...  ...  ...  ...  345 

Preservation  and  examination  of  ...  ...  ...  ...  ...  479 

Chcetx,  definition  of    ...          ...         ...  ...  ...  ...  ...  ...  165 

Chaatotaxy,  definition  of         ...          ...  ...  ...  ...  ...  ...  165 

Chagas,  on  Trypanosoma  cruzi         ...  ...  ...  ...  ...  ...  114 

Charts,  for  representing  geographical   and  topographical  distribution  of 

disease,  method  of  drawing        ...  ...  ...  ...  ...  ...  476 

Value  of        ...         ...          ...  ...  ..  ...  ...  476 

In  blackwater  fever  and  yellow  fever,  value  of  ...  ...  ...  470 

In  representing  secretion  rates  of  substances,  value  of  ...  ...  475 

In  secretion  rates  of  substances,  method  of  using  ...  ..  ...  475 

Chelicerre,  in  acarina  ...          ...          ...  ...  ...  ...  ...  297 

Chemical  products  of  organisms        ...  ...  ...  ...  ...  ...  406 

Indol  formation   ...          ...          ...  ..  ...  ...  ...  406 

32 


498  INDEX 

PAGE 

Chemicals  suitable  for  treatment  of  cover-glasses  before  use         ...  ...        17 

Chigoe  {see  Sarcopsylla  penetrans). 

Chinese,  Meckel's  diverticulum  prevalent  among  ...         ...  ...  ...       23 

Chironomidit,  character  of     ...         ...         ...         ...  ...         ...         167,  169 

Chlorides,  in  water,  test  for,  qualitative      ...         ...  ...         ...  ...     433 

Chloroform  and  benzol  used  for  estimating  specific  gravity  of  blood       ...      142 
Chlorosis  ...         ...         ...  ...         ...  ...         ...         ...         ...     442 

Changes  of  red  blood-corpuscles  in       ...         ...  ...         ...  ...       61 

Christophers  : — 

Demonstration    of  developmental    forms   of    I  iroplasma   cam's   in 

Rhipicephalus  sanguineus  ...  ...  ...  ...  ...  ...      1 04 

Development  of  Hcemogregarina  cam's...  ...  ...  ...  ..      106 

Mammalian  hremogregarine  described  by        ...         ...         ...  ...      106 

Chromatin  :  — 

In  crescents  ...  ...  ...  ...  ...  ...  ...  ...       94 

Changes  in  arrangement     ...         ...  ...         ...  ...  ...       95 

Modification  of  Romanowsky  stain  for  ...  ...  ...  ...       66 

Staining  of  ...         ...         ...         ...         ...         ...         ...         ..        68 

Chrysomyia,  characters  of      ...  ...  ...  ...  ...  ...  ...      194 

Chrysomyia  mace/iaria,  geographical  distribution...  ...  ...  ...      194 

Chrysops,  characters  of  ...  ...  ...  ...  ...  ...  ...      175 

Cicada,  characters  of  ...  ...  ...  ...  ...  ...  ...  ...      157 

Cigarette  paper  method  of  preparing  blood-films    ...         ...         ...         ...       50 

Cimex  ciliatus  295 

Columbarius         ...  ...  ...  ...  ...  ...  ...  ...     295 

Hirundinus  ...         ...         ...         ...         ...         ...  ...  ...     295 

Inodorus    ...         ...         ...         ...         ...         ...         ...         ...         ...     295 

Lectuarius  ...  ...  ...  ...  ...  ...  ...         294,  295 

Characters  of  294,  295 

Rotundatus  294,  295 

Characters  of  295 

CimicidcE  294 

Characters  of        ...     294 

Cladorchis         350 

Clark's  process  for  estimating  hardness  in  water    ...         ...         ...  434 

Climates  (temperate),  post-mortem  examinations  in,  differ  from  those  in 

Tropics  ...         ...         ...         ...         ...         ...         ...  ...       21 

Clinorrhyncha,  characters  of...         ...         ...         ...         ...         ...         ...     168 

Clonorchis         354,  356 

Cobb,  measurements  of  adult  filarke  131 

Coccidia  in  human  fceces        367 

Coccidia  32I"324 

Classification        322,  323 

Demonstration  by  staining         323 

Life  cycle 321,322 

Oocysts  of  322,323 

Cockroaches,  characters  of 156 

Coitus,  dourine  transmitted  by  112 


INDEX 

Coleoptera,  characters  of 

Metamorphosis  of 
Colitis,  membranous,  casts  of  rectum  in  faeces 
Collembola 

Colorimetric  estimations 
Compsomyia 

Afacellaria 
Condenser  (sub-stage),  for  laboratory  microscope  ... 
Congo  floor  maggot     ... 
Conorrhinus  sanguisuga         ...  ...  ...         "...  ... 

Supposed  carrier  of  Trypanosoma  cruzi 
Copepoda,  description  ... 

Carrier  of  disease 
Copper  in  water,  test  for,  qualitative  ...         ...     "    ... 

Cordylobia,  characters  of 

Anthropophaga 
Corethrina,  larvae  of,  anatomy 

Respiratory  tubes  in 
Corpuscles,  red,  see  Blood  corpuscles,  red  ;  white,  see  Leucocytes 
Cover-glasses    ... 

For  blood-examination   ... 

For  examination  of  fceces  ...         ... 

"  Squash  "  preparations... 

Preparation,  methods  of... 

Method  of  removal  of  oil  from  ... 

Treatment  with  oil  for  preservation     ... 
Crab-louse 
Craneflies 

Crenation  of  blood  corpuscles 
"  Crescents,"  chromatin  in     ... 

Examination  of  blood  for 

In  subtertian  (malignant)  malaria 
Cricket,  character  of  ...  ... 

Crustacea 

As  carriers  of  disease 
Cryptococcus,  parasite  in  horses  with  epizootic  lymphangitis 
Ctenocephalus  ... 

Serraiiceps  ...         ...••... 

See  also  Dog-flea. 
Ctenophthalmus 
Ctenopsylla  musculi    ... 
Ctenopsylla 
Culex    ... 

Characters  of 

Egg-laying  in  masses  or  rafts 

Egg  of       

Fatigans    ... 

Carrier  of  Filaria  nocturna 

Larvae  of,  respiratory  syphon  in 


9i 


229, 


499 

PAGE 

157 

'57. 

158 

331 

156 

447 

448 

194 

160 

8 

194 

295 

114 

309 

309 

428 

195 

195 

268 

269 

268 

116 

18 

18 

18 

1 

7,  50 

17 

16 

293 

167 

45 

94 

253 

,  92 

.  252 

156 

155 

309 

123 

289 

288 

,  290 

289 

290 

289 

J3° 

228 

261 

263 

233 

.  253 

256 

271 

5°° 


INDEX 


I-AGE 


Cu  lex —continued. 

Pipiens 

Characters  of 
CulicidiC 

Breeding  places  of 

Characters  of 

Larvae  of,  anatomy 

Respiratory  syphon  in 
Respiratory  tubes  in 
Syphonate     ... 

Pupa?  of,  respiratory  tubes 

See  also  Mosquitoes. 
Culicina  :  — 

Classification  of   ... 

Egg-laying  in  masses  or  rafts     ... 

Feeding  time 

Genera  of... 
Culicina: 
Cultures  must  be  made  from  fresh  specimens 

Preservation  in  laboratories 
Curare,  arrow  poison  .. 
Curnpira 

Cyclops,  fresh  water     ... 
Cyclorrhapha  : — 

Characters  of 

Classification 


Daddy-long-legs 

Dance-flies,  characters  of 
Darling  : — 

Method  of  oxygenating  water  fordoreeding  larvae  of  mosquitoes 
On  mosquitoes 
Daylight,  good,  essential  for  laboratories  in  Tropics 
Degeneration  in  tissues 

"  Cloudy  swelling." 
And  pigment  deposits 

See  also  Amyloid,  Fatty  and'Fibrous  Degeneration. 
Dcinocerites 

Delhi  boil  

Parasites  causing 
Demodex  follicular  u  m 
Demodicidce 
Dendromyia 
Dendromyinu  ... 
Dengue  fever    ... 
Dennatocentor,  Genus  of  Rhipicephala 

Reticulalus,  carrier  of  Firoplasma  cam's  in  Europe 
Desvoidea,  larvw  of,  respiratory  syphon  in  ...  


I02 

226, 

228 

234 

.56 

168 

268 

269, 

271 

268 

269 

279 

226 

26l 

159 

225 

234 

26 

5 

482 

169 

310 

167 

I  66; 

,  167 

164 

167 

274 

225 

1 

317-326 

3i7 

3i: 

[-320 

231 

328 

123 

308 

297 

,3°8 

232 

208 

>  232 

259 

306 

103 

271 

INDEX 

Desvoidy,  character  of  Hydrolira,  and  Hylemyia  .. 
"  Diarrhrca,  Tropical,"  often  dysenteric    ... 

Air-bubbles  and  gaseous  pigmentation  in 
Diazo-reaction  of  urine,  constant  in  typhoid  fever 
Dicroccclium 
Digenia 

Dinomimetcs    ... 
Diplococci 
Dip/era... 

Characters  of 

Classification  of   ... 

Metamorphosis  of 

Poison  of  ... 
Dipylidium  canimcm,  carrier  of 
Disease,  blood  changes  in 
Dog:- 

Blood  of,  filaria  in 

Definitive  host  of  Tcenia  echinococcus  ... 

Usual  host  of  Dipylidium 
Dog-flea,  carrier  of  Dipylidium  caninum   ... 
Dogs : — 

Heart  of,  Filaria  immitis  found  in 

Leucocytes  of,  hremogregarine  in     .    ... 

See  also  Jaundice,  Epidemic. 
Donkeys  and  horses,  Piroplasma  equi  produces  disease  only  in. 
Dourine  : — 

Animals  refractory  to 

Animals  susceptible  to    ... 

Geographical  distribution 

How  transmitted 
Dragon-fly,  characters  of 
Drigalski-Conradi's  medium... 
Dutton's  membrane  in  labium  of  mosquitoes 
Dysentery,  A?)iccba  coli  in 


501 

PACK 
195,   I96 

••■  334 
334,  335 
...     376 

354-356 

•••     354 

•     231 

...     390 

■»  159 
157,  161 
...     166 

157,  158 
■■■  159 
...     283 

57,  59 

124 
345 
35o 

283 

131 

105 

103 

"3 
"3 
112 
112 
156 
406 
258 
365 


Earwig,  characters  of 
Eggs  :— 

Of  mosquitoes 

Method  of  laying  in  different  genera 

Retention  of  vitality  

Of  parasites  in  feces 
Measurements 
Ehrlich-Biondi,  method  of  fixation  for  staining  by 
Emphysema  : — 

Of  intestines  produced  post-mortem  in  Tropics 
Of  liver  produced  post-mortem  in  Tropics 
Empodium,  definition  of 


'56 


273 

261 

262, 

263 

340, 

344 

344 

5i 

24 

24 

165 

502  INDEX 

PAGE 

Endemic  index  : — 

Charts  used  in     ...          ...          ...          ...          ...          ...          ...  465-471 

Methods  of  obtaining,  in  malaria  ...         ...      461,462,463,464,465 

Entamaba  coli...          ...          ...          ...                      ...          ...          ...  366,  367 

Hystolytica            ...          ...          ...          ...          ...          ...          ...  366,  367 

development  of        ...         ...         ...          ...         ...         ...  ...     367 

Enteric  fever,  see  Typhoid  fever. 

Entozoa,  examination  for  post-mortem,  in  Tropics             ...         ...  ...       24 

Eosin  : — 

Counter-staining  by         ...          ...          ...          ...          ...          ...  ...       52 

(Extra  B.A.,  Griibler's) 67 

Staining  of  parasites  in  blood  by           ...         ...         ...         ...  ...       62 

Of  red  corpuscles  by            ...          ...          ...          ...          ...  ...        53 

And  hematoxylin,  staining  with           ...          ...          ...          ...  ...       72 

Azur,  method  of  staining            ...          ...          ...          ...          ...  ...       68 

Eosinophile  leucocytes,  characteristics  of   ...         ...          ...          ...  ...        56 

Eosinophiles,  relative  proportion  of,  in  diseases     ...         ...         ...  ...       58 

Epipharynx  of  fleas     ...          ...          ...          ...          ...          ...          ...  ...     285 

Eugregarina   ...         ...         ...         ...         ...         ...         ...         .  .  ...     1 08 

Subdivisions  of    ...         ...         ...          ...          ...         ...          ...  ...      108 

Europe,  carrier  of  Piroplasma  cants  in        ...          ...         ...          ...  ...      103 

Europeans,  weight  of  brain  in,  age  at  which  maximum  is  attained  ...       22 
Weight  of  organs  of  body  in  health  and  disease  in,  vary  from  Tropical 

standard             ...          ...          ...          ...          ...          ...          ..  ...       22 

Eye-piece  micrometer  in  microscope  work  ...          ...          ...          ...  13,   14,  15 

F.eces  : — 

Amoeba  coli  found  in       ...         ...         ...         ...         ...         ...  365,396 

Analysis  of           337-339 

Colour  of 332-334 

Effect  of  abnormal  articles  on        ...         ...         ...         ...  332,333 

Effect  of  diet  on       332 

Effect  of  diet  on  bulk  of 335,  336 

Effect  of  disease  on  bulk  of       ...         ...          ...          ...         ...  336,337 

Embryos  of  parasites  passed      ...         ...         ...         ...         ...  ...     344 

Examination  of    ...          ...          ...          ...          ...          ...          ..  330-344 

Acid  or  alkaline  reaction    ...          ...          ...          ...         ...  ...     335 

Air-bubbles  and  gaseous  fermentation       ...          ...         ...  334,  335 

Bulk  of  feces            335-339 

Consistence  of  the  stool,  "looseness"     ...          ...          ...  ...     334 

For  bile  acids           ...         ...          ...         ...         ...         ...  ...     333 

For  bile  pigments    ...         ...         ...         ...         ...         ...  ...     333 

Macroscopic              ...         ...         ...         ...         ...          ...  33°-332 

For  mucus    ...         ...          ...         ...         ...         ...          ...  331,  332 

For  odour     ...         ...          ...         ...          ...         ...         ...  ...     335 

For  parasites            339-344 

For  presence  of  urobilin     ...         ...          ...         ...          ...  333>  334 

Parasites' eggs  in,  varieties  and  description    ...          ...         ...  340-344 


INDEX 


S°3 


Faeces — continued. 

Species  of  parasites  passed  naturally  and  after  anthelmintics 

Straining,  for  discovery  of  parasites 
Farrant's  solution 
FasciolidcB  (flukes),  eggs  of     ... 
Fasciola 

Hepalica,  life  history  of  .  . 
Fasciolelta 
Fasciolidic 

Structure  of 
Fasciolopsis 
Fatty  degeneration      ...  ...         ...         ... 

As  factor  in  Tropical  diseases 
Staining  for  ... 
Fats:  — 

Detection  of,  in  feces     ... 

Difference  in  digestibility  of 
Favus     ... 
Fever : — 

See  African  relapsing  fever  ;  Texas  fever. 
Fibrous  degeneration  ... 

Marchi's  method  of  demonstration 
Of  nerve  tissue 
Ficalbia 

Field-rat  (Indian),  hsemogregarine  parasitic  in       

Filaria,  embryos  of,  see  Microfilaria. 
Filaria  :-— 

Human 

In  blood  of  dog    ... 

In  mosquitoes,  demonstration    ... 

In  tissues  ... 
Filaria  bancrofli 

Found  in  lymphatic  system  of  man 

Head  of  female    ... 

In  urine  and  blood 

Tail  of  female 
Filaria  demarquayi     ... 

Embryo  of 

Head  of  female    ... 

Tail  of  female 
Filaria  immitis 

Carried  by  mosquitoes     ... 

Development  of  ... 

Found  in  heart  of  dogs    ... 
Filaria  loa 

Difficulty  of  extraction  from  human  body 

Subcutaneous  position  in  human  body... 
Filaria  magallnesi 

Medinensis 


344 
339 
250 

343 
354 
354 
354,  356 
35°,  352 

353,  354 

354,  356 
317-319 
•  ••  3i9 

3i7,  3i8 

338,  339 
•••  339 
...  417 


•••  319 
...  320 

319,  320 

...  232 
106,  107 


200 
124 
247 
328 
[29,  258 
130 
133 
372 
134 
130 
129 

135 
138 
200 
258 
259 
131 
129 

131 

!3° 

7i 
310 


be  made  in  fresh  worms 


504  INDEX 

Filar ia  noctuma  :  — 

Carried  by  mosquitoes  of  several  genera  and  species 

(  arriers  of 

Development  in  mosquitoes,  demonstration    ... 
Filar  ia  ozzardi ... 

Tail  ot  female 
Filaria  perslatis 

Embryo  of 

Head  of  female    ... 

Tail  of  female 
FilariiC,  calcified,  in  human  body     ... 
Filaria  : — 

Diptera  as  hosts  for         

Geographical  distribution  of  species     ... 

Habitat  in  body  of  species 

Adult,  description  of 
Measurements  of 
Measurements  should 
Recognition  of 

(Avian) 

Positions  in  body  of  birds 

Human 

Transmission  to  others  and  re-infection  of  individual 

Human,  adult,  measurements  of 

Resemblance  of  some  species  to  each  other,  close 
Transparent  cuticle  of 

Localities  of,  in  human  body 

Method  of  escape  from  mosquitoes 

Mounting  of,  in  glycerine 

Points  of  difference  and  resemblance  of  species  ...   133, 

Table  showing 

Searching  tissues  for,  background  for  ... 

Staining  of  specimens     ... 
Filaiiasis,  carriers  of   ... 

Increase  of  eosinophiles  in 
Fish,  blood-plasma  of  trypanosomes  in 
Fixation  of  paraffin  sections  on  slide 

Of  films,  methods  of 

Of  issues,  time  required  for 
Flagella  or  motile  organism  ... 

Staining     ... 
Flagellataor  Mastigophora,  Leishman-Donovan  bodies  assigned 
Flagellates        ...         ... 

See  also  Leishman-Donovan  bodies. 
Flea,  characters  of, 

Feeding  time 

(Jigger)      

Fleas 

Anatomy,  external 

Internal         


34 


PAGE 

256 
229 
256 
129,  130 
134 
>30 
129 

135 

138 
130 

...  161 
136,  137 
136,  137 
131 
131 
132 
131 
259 
131 
258 

2SS 
132 
132 
132 
130 
257,  258 
...   138 

135.  138 

136,  137 
...  131 
126,  127 

...   23O 

5S 


...  51 
28,  29 

...  393 
394 
122 

326-328 

157 
'59 
160 
283 
284 
287 


INDEX  505 

Fleas—  continued.  PAGE 

Antenna  of           ...          ...          ...          ...          ...  ...  ...  ...  285 

Capture  and  examination  of      ...          ...          ...  ...  ...  ...  283 

Carriers  of  Bacillus  peslis           ...          ...          ...  ...  ...  ...  283 

Classification        ...          ...          ..           ...          ...  ...  ...  ...  287 

Dissection...          ...          ...          ...          ...          ...  ...  ...  ...  287 

Epipharynx  of      ...          ...          ...          ...          ...  ...  ...  ...  285 

"Gizzard"  of       287 

Larva?  of  ...          ...          ...         ...         ...          ...  ...  ...  ...  286 

Mandibles  of        285 

Maxillae 285 

Metamorphosis  of            ..           ..           ...          ...  ...  ...  ...  286 

Mounting  of         ...          ...          ...          ...          ...  ...  ..  ...  284 

Thoracic  segments  of      ...          ...          ...          ...  ...  ...  ..  285 

See  also  Pulicidce,  Sarcopsyllidic,  Vermipsyllidtc . 

von  Fleischl's  hsemometer      ...          ...         ...          ...  ...  448,449,450 

]- lemming's  mixture,  fixation  of  tissues  by  ...          ...  ...  ...  ...  29 

Flukes,  eggs  of  342,343 

See  also  Trematodes        ...          ...          ...          ...  ...  ...  ...  350 

Flushing  off  stains,  method  of           ...          ...          ..  ...  ...  ...  52 

Food  for  mosquitoes    ...          ...          ...          ...          ...  ...  ...  ...  282 

Ol  larvce  of  mosquitoes   ...          ...          ...          ...  ...  ...  ...  274 

Forest-fly          ...         ...         ...         ...          ...          ...  ...  ...  ...  195 

Formaldehyde,  method  of  fixation   ...          ...          ...  ...  ...  ...  49 

Formalin,  method  of  fixation              ...          ...          ..  ...  ...  ...  51 

First  bath  of,  for  museum  preparations            ...  ...  ...  ...  25 

Formol  alcohol,  for  fixation  and  hardening  of  tissues  ...  ...  ...  28 

For  rapid  fixation  of  tissues           ...          ...  ...  ...  ...  28 

Freezing  microtomes,  descriptions  of           ...         ...  ...  ...             34,  35 

Frons,  definition  of     ...         ...          ...          ...          ...  ...  ...  ...  162 

Fungi,  Gram's  method  of  staining    ...         ...          ...  ...  ...  ...  41S 

In  interior  of  body          ...         ...          ...          ...  ...  ...  ...  419 

Tropical,  attacking  skin  and  hair          ...          ...  ...  ...  ....  417 


Gadfly,  characters  of           ...  ...  ...  ...  ...  ...  ...  174 

Feeding  time        ...         ...  ...  ...  ...  ...  ...  ...  J-59 

Gall  midges,  characters  of      ...  ...  ...  ...  ...  ...  ..  168 

Gamasidse         297 

Gametes,  union  to  form  zygotes  ...  ...  ...  ...  ...  ...  109 

Gametocytes     ...         ...         ...  ...  ...  ...  ...  ...  76.  252 

Development  of   ...          ...  ...  ...  ...  ...  ...  ...  80 

Genesis  of...          ...          ...  ...  ...  ...  ...  ...  ...  96 

Proportion  forming  zygotes  ...  ...  ...  ...  ...  ...  254 

Shape  in  benign  tertian  and  quartan  malaria  ...  ...  ...  ...  91 

Staining  of            ...          ...  ...  ...  ...  ...  ...  93,  94 

Gas  formation  in  bacteria       ...  ...  ...  ...  ...  ...  ...  404 

Gast  iodise  its  kominis   ...          ...  ...  ...  ...  ...  ...  ...  35^ 

Gastrophilus  equi        ...         ...  ...  ...  ...  ...  ...  ■■■  160 

Gena-,  definition  of     ...         ...  ...  ...  ...  .  .  ...  ...  162 


148 
i48 
167 
166 
112 
116 
138 
39 
135 
142 


506  INDEX 

PAGB 
Genital  organs  of  mosquitoes  ...  ...  ...  ...  ...  ...      251 

Geographical  distribution  of  dourine  ...  ...  ...         ...  112 

Of  pedicuUv,  variation  under     ...  ...  ...  ...  ...     293 

Of  Pulex  cheopis  289 

Of  Satcopsylla  penetrans (Jigger  or  Chigoe)     ...  ...  ...  ...     291 

Of  Schistosoma    ...  ...         ...         ...  ...  ...         ..  371 

Of  Screw-worm  fly  ...  ...  ...  ...  ...  ...  ...      194 

Of  Slreptotlirix  madura  ...  ...  ...  ...  ...  ...     412 

Of  Surra  ...         ...         ...         ...         ...         ...         ...         ...      112 

Gerbillus  (indicus),  see  Field-rat  (Indian). 

Giemsa's  method  of  staining  67,118,123 

Gizzard,  of  fleas  ...  ...  ...  ...  ...  ...  ...  ...     287 

Glass  tubes  (Wright's)  for  obtaining  and  diluting  blood-serum 

Description  of 
Glossina,  characters  of 

Larva  of 
Glossina  vtorsitans,  Trypanosoma  brucei carried  from  animal  to  animal  by 
Glossina  palpalis,  carrier  of  human  trypanosome 
Glycerine,  mounting  of  filarioe  in 

And  albumin  method  of  fixation 

And  alcohol,  examination  of  nematodes  in     ...         ...         ...         134. 

And  water  used  for  estimating  specific  gravity  of  blood 

And  water  and  potassium  acetate,  preservation  of  museum  prepara- 
tions in  ... 

Broth         

Method  for  mounting  mosquitoes 
Gmelin's  reaction 
Gnats,  characters  of    ... 

(Buffalo) 

(Turkey)    .  . 
Goadby,  K.  W. ,  ages  at  which  teeth  are  cut  in  Europeans 
Gower's  h;emoglobinometer  ... 

Solution    ... 
Grabhamia  dorsalis,  eggs  of,  method  of  laying 

Retention  of  vitality 
Gram's  method  of  staining     ... 

Micro-organisms,  retaining  stain  when  treated  by 
Not  retaining  stain  when  treated  by 
Granules  present  in  myelocytes 

Revealed  by  Leishman's  stain 
Granuloma  (fungating),  Spirochcrta  pertenuis  present  in 
Grasshopper,  characters  of    .. 
Gray,  Douglas,  observations  on  blood  counts 
Gregarines 

Body-form  well  defined,  not  amceboid 

Development  of ... 

Method  of  staining 

Reproduction,  method  of 

Sub-orders  of 


235. 


,26 

383 
236 

333 

167 

172 

...     172 

...     456 

448,  449 

...     150 

...     261 

263 

•■■  395 
422,  423 
...  424 
...  70 
...  70 
119 
156 
...  446 
...  107 
...     109 

107,  108 

■     479 

108,  109 
...      108 


INDEX  5°7 

l'AG  E 

Guinea-worm  ...         ...         ...  ...         ...         ...  ...         ■••         •••     3'° 

Gutta-percha  method  of  preparing  blood-films       ...  ...  ...  •••        5° 

Gutters,  badly  graded,  as  artificial  breeding-places  for  mosquito  larve  ...      278 


HiP.magogus 

1 1  remamoebe,  differences  between  piroplasmata  and 
Hemamoebide  not  found  in  blood-plasma  ... 
Htvmaphysalis,  genus  of  Rhipicephale 

Leachi,  carrier  of  Piroplasma  cants  in  South  Africa  ... 
Causing  canine  piroplasmosis  ... 
Hcematopinus 

Ilematopota,  characters  of   ... 
Hematoxylin,  staining  of  microfilarias  with 

Staining  of  parasites  in  blood,  by 

Staining  with 

And  eosin  staining  with 
Hematuria,  distinguished  from  hemoglobinuria  ... 

From  bilharzia  infection 

From  filariasis 
H<emocyslidinm  (pigmented  parasite)  in  snakes 
Hemocytometer,  graduated  pipette  of,  for  dilution  of  blood-serum 

Thoma-Zeiss 
Hemoglobin,  altered  source  of  pigment  deposits  ... 

Colouring  matter  of  blood 

Dissolution  in  blood-serum  (laking  of  blood)  ... 

In  blood-serum,  determination  of  presence  or  absence 
(Diluted)  medium  for  cultivation  of  trypanosomes 

Liberation  of,  by  staining 

Reduced,  spectra  of 

Removal  from  red  corpuscles    ... 

Solution  of 

Scale,  Tallquist's 
Hemoglobinometer,  Gower's 

Hemoglobinuria,  characteristic  of  "  blackwater  fever  "... 
Ilcemogregarina  :  — 

Description  of 

Occur  commonly  in  blood  of  cold-blooded  vertebrates 
In  blood  of  mammals 

Unpigmented  parasites  ... 
Hamogregarina  balfouri 

Canis 

Development  of 
Sexual  ... 

Funambuli 

Gerbilli 
Hemogregarines 

In  blood-plasma  ... 


102, 

103 

no 

305 

103 

303- 

304 

292 

175 

127 

62 

52, 5; 

[>  72 

72 

371 

371 

372 

102 

148 

438, 

439 

3" 

144 

146 

143 

152 

47 

143 

144 

144. 

145 

450 

448 

.449 

372 

105 

105 

105 

105 

107 

i°5 

106 

106 

,  107 

107 

107 

105 

no 

;o8 


INDEX 


PACE 

Hemolysins     ...         ...         ...         ...         ...         ...         ...         ...  ...     150 

Hemolysis : — 

In  anemia,  causes  of      ...          ...          ...          ...         ...         ...  443 

Source  of  pigment  deposits        ...         ...          ...         ...         ...  ...     3"5 

Hcemometer,  von  Fleischl's  ...  ...         ...         ...         ...  448,449,450 

Hemosporidia,  development  of,  general  summary            ...         ...  ...       76 

Hair,  tropical  fungi  attacking            ...          ...          ...          ...          ...  ...     417 

Ilalteridium  : — 

In  birds 100,  101 

Sexual  phases  of  ...          ...         ...         ...         ...          ...         ...  ...     101 

Hardness  in  water  :  — 

Cause  of    ...          ...          ...          ...          ...          ...          ...  ...     434 

Method  of  estimating     ...          ...         ...         ...         ...         ...  ...     434 

Harpagomyia  ...          ...          ...          ...          ...          ...          ..           ...  ...     232 

I Iearson's  incubator    ...          ...          ...          ...          ...          ...          ...  19)  20 

Heart,  dilatation,  in  dogs,  from  presence  of  filaria  in  large  numbers  ...     124 

Heartvvater  of  sheep  and  goats,  caused  by  A>nblyomma  hebricum  ...     304 

Heat,  fixation  by,  to  be  avoided       ...          ...         ...         ...         ...  ...       51 

Helminths,  in  tissues,  staining  for  eggs  and  larve  of        ...         ...  ...     329 

Hemiptera         293,  294 

Adult  development  of     ...         ...          ...         ...          ...           ..  ...     158 

Characters  of       ...          ...          ...          ...          ...          ...          ...  ...      157 

Hermann's  solution,  fixation  of  tissues  by  ...          ...         ...         ...  ...       29 

Herpetomoiias  ...          ...          ...          ...          ...          ...          ...          ...  ...      122 

Helerophyes      ...         ..           ...         ...         ...         ...         ...         ...  ...     35^ 

Hexapoda  (see  Insecta). 

Hippobosca  equina       ...          ...          ...          ...          ...          ...          ...  ...      199 

Kufipes,  probably  carrier  of  Trypanosoma  theileri    ...         ...  ...     113 

Hippoboscidii,  characters  of  ...          ...          ...          ...          ...          ...  167,197 

Hodgesia  232 

Homalomyta,  characters  of    ...          ...          ...          ...          ...          ...  195,196 

Horder's  method  of  blood  examination       ...          ...          ...          ...  ...       44 

Horse,   disease   resembling  "  Nagana "  produced  in,   by    Trypanosoma 

dimorphon         ...          ...          ...          ...          ...          ...          ...  ...      114 

Horse-flies,  character  of         ...          ...         ...           ..          ...         ...  167,174 

Horses  :  — 

Epizootic  lymphangitis  in           ...          ...         ...          ...         ...  ...     123 

"  Mai  de  Caderas,"  disease  of  ...          ...         ...          ...         ...  ...     113 

Surra  fatal  to        ...          ...          ...          ...          ...          ...          ...  ...      112 

"  Verminous  aneurisms  "  in      ...          ...         ...         ...         ...  ...     124 

And  donkeys,  Piroplasma  eqni  produces  disease  only  in       ...  ...     103 

House-fly,  metamorphosis  of             ...          ...          ...          ...          ...  ...      157 

Howard,  Dr.,  report  by,  as  regards  prophylaxis  in  malaria          ...  472,  473 

Iluppert's  test 333 

Hyaline  cells  of  leucocytes     ...          ...          ...          ...          ...          ...  ...       55 

Ilya/omma,  genus  of  Ixodimv            ...          ...          ...          ...          ...  ...     305 

Hyalomma  agyplicnm,  developmental  form  of  Piroplasma  bigeminum  in     104 

Hydrotaa  (Desvoidy),  characters  of             ...          ...          ...          ...  195,196 

Hylemyia  (Desvoidy),  characters  of            I95>  196.  197 


INDEX 


5°9 


Hymenoptera  :  — 

Characters  of 

Metamorphosis  of 
Hyphai 

Aerial 

Sub-aerial 
llyphomycetes 

See  also  Moulds. 
Hypopharynx  of  mosquitoes. 
Hystrichopsylla 

Talpx       I 


...  156 
157,  158 
...  418 
...  418 
...  418 
...  417 


2*8, 


239 
289 
290 


Ice  and  salt  freezing  mixture             ...         ...          ...         ...          ...  ...       35 

Illuminating  apparatus  for  laboratory  microscope  ...         ...         ...  ...         S 

Imago : — 

And  metamorphosis         ...          ...          ...          ...          ...          ...  ...      158 

Subsequent  distribution  by,  of  bacteria  imbibed  by  larva     ...  ...     260 

Imbedding  of  fixed  tissues,  celloidin  method          ...          ...          ...  ...       33 

Paraffin  method            ...          ...          ...          ...          ...  ...30-32 

Incubator  (Hearson's)...          ...          ...         ...          ...         ...          ...  19,  20 

Incubators  for  laboratories     ...          ...          ...          ...          ...          ...  ...          4 

India,  carrier  of  Piroplasma  cants  in             ...          ...          ...          ...  ...      103 

India-rubber  teats  (Wright's)  for  drawing  up  fluid  into  mixing  chamber 

149,  150 

Indian  ink  method  of  demonstrating  Spirochccta  pallida  ...          ...  ...      120 

Indican,  how  best  detected  in  urine             ...         ..           ...         ...  ...     374 

Indol  formation,  chemical  product  of  bacteria       ...         ...         ...  ...     406 

Infection,  diptera  as  carriers  of         ...         ...         ...         ...         ...  ...     160 

Infusoria             ..          ...          ...          ...          ...          ...          ...          ...  ...     369 

Inoculations  for  bacteriological  examination  of  water      ...          ...  ...     414 

Insecta,  characters  of...         ...          ...         ...         ...          ...          ...  ...     155 

Intestines : — 

Abnormal  appearances  in,  at  post-mortem  examinations  in  Tropics       23 

Emphysema  of,  in  patches,  produced  post-mortem  in  Tropics  ...       24 

Examination  for  entozoa  post-mortem  ...          ...          ...          ...  ...       24 

Gaseous  distension  produced  post-mortem  in  Tropics...          ...  ...       24 

Iron  : — 

In  water,  test  for,  qualitative    ...         ...          ...         ...          ...  ...     426 

Quantitative             ...          ...         ...         ...  ...     427 

Irrigation-systems  as  artificial  breeding  places  of  mosquito-larvss  ...     278 

Ixodie    ...         ...         ...         ...         ...         ...         ...         ...         ...  305,  306 

Ixodes,  genus  of  Ixodituc        ...          ...          ...          ...          ...          ...  ...     305 

Ixodidic,  sub- families              ...          ...          ...          ...          ...         ...  ...     297 

Ixodince            ...         ...         ...         ...         ...         ...         ...         ...  297-306 


/acnlus  goudoni  {see  Jerboa). 
Jalousies,  for  protection  of  laboratories 


5io 


INDEX 


Jaundice:  — 

Epidemic  of  dogs,  cannot  be  reproduced  in  other  animals  ...  ...      103 

Parasite  causing       ...         ...         ...          ...          ...          ...  ...      103 

Hematogenous    ...         ...          ...         ...          ...         ...          ...  ...      146 

Occurring  in  Tropics      ...         ...         ...          ...         ...         ...  ...     373 

Jerboa  (faatlus  gottdoni),  hxmogregarine  in  red  blood-corpuscles  of  ...      106 

Jigger  flea         ...         ...          ...         ...         ...          ...         ...          ...  ...      160 

See  also  Sarcopsylla  penetrans, 

foblotinu-           ...          ...          ...          ...          ...          ...          ...          ...  ...     208 

Kaiserlixg's  method  of  preserving  museum  preparations          ...  ...       25 

Kala-azar : — 

Due  to  Leishman-Donovan  bodies       ...          ...          ...             122,  123,  328 

Leucocyte  variation  in    ...          ...          ...          ...          ...         ...  57>  59 

Kerosene  Smokeless  Burner,  "  Primus "     ...         ...         ...          ...  ...         4 

Klang,  experiments  in  malaria  at      ...         ...         ..           ...         ...  470-472 

Koch  :   Descripiion  of  developmental  form  of  Piroplasma  bigeminam  in 

Rhipicephaliis  australis,  R.  evertsi  and  Hyalotnma  iVgypticnm  ...      104 
Koch's  : — 

Comma  bacillus     ..         ...         ...         ...         ...         ...         ...  ...     416 

Postulates  as  to  pathogenicity  of  organisms    ...          ...         ...  ...     411 

Steam  sterilizer    ...          ...         ...          ...         ...          ...         ...  17,  18 


Labium  of  mosquitoes 
Laboratories  : — 

In  iropics,  "  burners  "  for 

Construction  of    ... 

Distilled  water  for 

Good  light  essential 

Incubators  for 

Lamps  for  work  at  night 

Microscopes  for   ... 

Preservation  of  cultures  in 

Protection  by  jalousies    ... 

Shelter  from  wind  and  dust 

Shelves  for 

Tables  suitable  for 

Water-tank  for 
Labrum-epipharynx  of  mosquitoes  ... 
Lakes,  pools  on  shores  of,  as  breeding-places  for  mosquito-larvce 
Lamblia,  characteristics  of     ... 

Symptoms 
Lamps  for  work  at  night  in  laboratories 
Larva,  bacteria  imbibed  by,  subsequent  distribution  by  imago    ... 

And  metamorphosis 
Larvae  of  fleas  ... 

Of  mosquitoes      ...         ...         ...         ...         ...  ...         •• 

Parasitic    ... 


237,  239 

3-  4 
1 

4 

1 

4 

5 

5-6 

5 

2 
1 


-.  j 
237.  239 
277 
368 

369 

5 

260 

157 
286 
264,  273 
...     160 


INDEX  511 

PACK 

Laveran,  nature  of  Leishman-Donovan  bodies       ...          ...          ...  ...     122 

Lead  in  water,  test  for,  qualitative  ...         ...         ...          ...          ...  ...     427 

Test  for,  quantitative      ...          ...         ...         ...         ...          •••  .-•     427 

Leishman-Donovan  bodies    ...         ...         ...         ...         ...         ...  71,  120 

Cause  of  kala-azar           122,328 

Classification  of  ...          ...          ...          ...          ...          ...          ...  ...      122 

Distribution  in  body        ...          ...          ...          ...          ...          ...  ...     327 

Method  of  obtaining  from  body            ...         ...         ...          ...  I2G,  121 

Nature  of 121 

Present  in  blood                ...          ...          ...          ...          ...          ...  ...      120 

Present  in  spleen,  liver,  and  other  regions     ...         ...          ...  ...      120 

Similarity  of  parasites  causing  Delhi  boil  to  ...          ...          ...  ...      123 

Of  parasites  causing  epizootic  lymphangitis  in  horses  to...     123 

Staining  of           ...          ...         ..           ...          ...          ...         ...  ...     121 

Staining  for          ...         ...         ..           ...          ...         ...         ...  327,  328 

Leishman's  modification  of  Romanowsky's  method  of  staining  ...  63,  64,  74, 

75.  78,  89,94,95,  118,  123 

Granules  in  myelocytes  revealed  by     ...          ...          ...         ...  ...       70 

Method  of  use 63,64,65 

For  trypanosomes            ...          ...         ...          ...         ...          ...  ...     115 

Leishman,  Sir  W.  B.,  nature  of  Leishman-Donovan  bodies        ...  122 

Lenses,  of  laboratory  microscope                  ...          ...         ...         ...  9,  12,  13 

[.epidopte}^,  characters  of      ...          ...         ...          ..           ..,         ...  ...     157 

Metamorphosis  of            ...          ..           ...         ...         ...          ...  157,  158 

Larvae  of  Tachinidre  parasitic  in           ...          ...         ...         ...  ...     197 

Lepidoselaga,  characters  of  ...          ...         ...          ...         ...         ...  ...     175 

Lepra  bacillus  ...         ...         ...         ...         ...         ...          ...          ...  ...     399 

In  mucus  discharged  from  nose             ...         ...         ...          ...  ...     399 

Leprosy,  amyloid  degeneration  in                 ...         ...         ...         ...  ...     319 

Leptothrix                     ...         ...         ...         ...         ...         ...         ...  ...     391 

Leucocyte  count,  differential              ...         ...         ...         ...         ...  ...       58 

Leucocytes,  abnormal  cells  resembling                     ...         ...         ...  ...       60 

Destruction  of  pathogenic  micro-organisms  by,  prepared  by  blood- 
serum                 ...         ...         ...         ...          ...         ...         ...  ...     152 

Diluted  blood  used  for  counting            ...         ...         ...          ...  ...      150 

Enumeration  of,  actual  reasons  for        ...         ...         ...         ...  58,  59 

Eosinophile,  coarse  granules  of             ...         ...         ...          ...  ...       56 

Distinguished  from  myelocytes              ...         ...         ...         ...  ...       61 

Granular,  characteristics  of        ...          ...          ...          ...          ...  ...       46 

Increase  of,  in  malaria                ...          .,.         ...          ...          ...  ...       57 

Increase  and  decrease  in  disease           ...         ...         ...         ...  ...     444 

Mononuclear,  large,  staining  of             ...          ...          ...          ...  55,  69 

Nuclei,  staining  of           ...          ...          ...          ...          ...          ...  ...        56 

Polymorphonuclear,  characteristics  of ...         ...         ...          ...  ...       56 

Relative  proportions  of,  in  diseases      ...          ...         ...         ...  ...       59 

In  normal  blood       ...          ..           ...          ...          ...          ...  ...        56 

Small,  staining  of             ...          ...          ...          ...          ...          ...  ...        55 

Staining  of            ...         ...          ...         ...         ...         ...         ...  ...       69 

Various  forms  of              ...         ...         ...         ...         ...         ...  54,  55 


512  INDEX 

PAGE 

Leucocythemia            ...          ...         ...         ...          ...  ...  ...  6l,  62 

Changes  of  red  blood-corpuscles  in      ...          ...  ...  ...  ...       62 

Mononuclear  myelocytes  in        ...          ...          ...  ...  ...  ...       60 

Leucocytosis,  importance  of,  in  disease       ...          ...  ...  ...  ...     443 

Leucocytozoon  canis  (see  Hamogregarina  canis). 

Leucopenia      ...         ...         ...         ...         ...         ...  ...  ...  ...     443 

In  malaria             ...          ...          ...          ...          ...  ...  ...  ...        57 

Levaditi's  method  of  staining  spirochetes  ...         ...  ...  ...  ...     479 

Li  mains            ...         ...         ..           ...         ...         ...  ...  ...  208,  231 

LinquatuliiLc   ...          ..           ..           ...          ...          ...  ...  ...  308,  309 

Lip-plate,  inferior,  in  larva.-  of  mosquitoes  ...          ...  ...  ...  ...     269 

Liver  abscess,  leucocyte  variation  in            ...          ...  ...  ...  ...       59 

Liver : — 

Aspiration  of,  to  obtain  Leishman-Donovan  bodies  ...  ...  ...     121 

Emphysema  of,  produced  post  tnorlem  in  Tropics  ...  ...  ...       24 

Leishman-Donovan  bodies  in  large  numbers,  present  in  ...  ...      120 

"  Looseness  "  of  stools,  important  in  tropical  practice  ...  ...  ...     334 

Louis  Jonner  stain                    ...          ...          ...          ...  ...  62,69,74,75 

methods  of  use             ...          ...          ...  ...  ...  62,63 

Lucilia,  in  cutaneous  myiasis            ...         ...         ...  ...  ...  ...     160 

Lungs : — 

Deeply  fissured,  of  negroes        ...          ...          ...  ...  ...  ...       23 

Leishman-Donovan  bodies  in    ...          ...          ...  ...  ...  ...     120 

Location  of  Microfilaria  bancrofli  in,  during  absence  from  peri- 
pheral blood             ...         ...                      ...  ...  ...  ...     129 

Lunula,  definition  of  ..            ...          ...          ...          ...  ,..  ...  ...      163 

Lymphangitis  (epizootic)  in  horses,  parasite  causing  ...  ...  ...     123 

Lymphatic  glands  :  — 

Leishman-Donovan  bodies  in    ...          ...          ...  ...  ...  ...     120 

Superficial  puncture  of,  to  obtain  Leishman-Donovan  bodies  ...     121 

Lymphatic  system,  human,  Filaria  bancrofti  found  in  ...  ...  ...      130 

Lymphocytes  : — 

Increase  of,  in  scurvy      ...          ...          ...           ..  ...  ...  ...       58 

Staining  of           ...         ...          ...          ...          ...  ...  ...  55,  69 

Lysol,  removal  of  oil  from  cover-glasses  by           ...  ...  ...  ...        17 


Mackogametes         ...         ...         ...  ...  ...  ...  ...  ...       93 

Madura  foot      ...          ...          ...          ...  ...  ...  ...  ...  ...     412 

Cause  of    ...          ...         ...         ...  ...  ...  ...  ...  ...     419 

Characteristics  of. ..          ...          ...  ...  ...  ...  ...  ...     412 

Maggot  (Congo  floor)...          ...          ...  ...  ...  ...  ...  ...      194 

Malaria  :  — 

Acute,  causes  of  death  in            ...  ...  ...  ...  ...  83,87 

Age  incidence  in  ...         ...         ...  ...  ...  ...  ...  ...     455 

Blood  count  in     ...          ...          ...  ...  ...  ...  ...  ...     442 

Case  mortality  in...          ...          ...  ...  ...  ...  ...  ...     457 

In  Central  Africa,  results  of  experiments  in    ...  ...  ...  472,473 

Changes  of  red  blood  corpuscles  in  ...  ...  ...  ...  ...       61 


INDEX 

Malaria— continued. 

Charts  used  in 

Effect  on  general  health... 

Endemic  index  in 

History  of 

Human,  parasites  allied  to  those  of,  found  in  animals 

Immunity  in 

Interval  between  relapses  in 

In  Klang,  results  of  experiments 

Liability  to  infection  in  ... 

To  relapse  in 
Melanin  deposits  in 
Pigment  in    ... 
Mortality  from     ... 
Parasites  of 

Activity  of  amoeboid  movement    ...  ..." 

Causing,  class  of 

Conveyed  by  diptera 

Definitive  host 

Distinctive  points  for  division  into  species 

Fragmentation  of  nucleolus 

Growth  ...         ...  ..  ...         ...       "... 

Human  blood-corpuscle  containing,  how  affected 
Formation  of  chromatin  nodules 
Found  in  blood-vessels,  staining  of  ... 

Most  commonly  post  mortem  in  brain  ... 

Staining  of 
In  tissues,  staining  of 
Phase  of 

Length  of  cycle  in 
Number  of  spores  in    ... 

Stained  specimens,  fallacious  appearances  ... 
Staining  of 

Selective  site  for  sporulation... 
Intermediate  host    ... 
Mosquito-phase 
Staining  of    ... 
Zoological  position  of 
Period  of  incubation  in  ... 

Process  due  to,  in  blood-vessels,  wrongly  described  as  thrombosis 
Prophylactic  measures  in,  value  of 
Quartan,  parasite  of,  characters 

Parasites  of,  selective  site  for  sporulation 
Parasite  of,  phases  in  asexual  and  sexual  development... 
Remote  or  indirect  mortality  in 
Seasonal  variation  in 
Species  of  Anophelinrc  in 
Spleen  te>t  in 
Statistics  concerning 

Dealing  with,  as  regards  population 

33 


5' 3 

I'AGE 
466-471 

•••  457 
460-465 
...  458 
102 
45S,  459 
•■•  4S5 
470-472 
•••  455 
•■•  455 
3U-3I6 
...       88 

•••     457 

...  77 
...  81 
...  71 
161 
...  77 
80,81 
...   79 

78,79 

88,  89 

...   80 

...   87 

83,  84,  85 

84,85 

...   86 

...   77 

...   81 

...   81 

...   98 

...   79 

82,  83 

...   77 

...   77 

47*  250 

...  109 

458 

87 

469 

97 

82 

89 

457 

459 

460 

463 

453 
454 


-^52 

9' 

•  92 

97 

Si 

92 

82 

97 

90 

90 

9i 

45S 

H5, 

334 

57 

.  59 

3'3 

3.6 

514  INDEX 

1  AGE 

M  alaria — continued. 

(Sub-tertian)  "  crescent  "  in 
Malignant,  "crescents"  in 
Parasites  of,  characters 

Length  of  cycle  difficult  to  determine  ... 
Phases  in  asexual  and  sexual  development 
Parasites,  selective  site  for  sporulation 
Tertian,  benign,  parasites  of,  characters 

Phases  in  asexual  and  sexual  development 
Malignant,  parasite  of,  effect  on  blood-corpuscles 
And  quartan,  shape  of  gametocyte  in 
Tolerance  in 
Urobilin  in  freces  in 
Variations  in  leucocytes  in 
Yellow  pigment  deposit  in 
Malarial  blood  : — 

Preparation  of  films  of   ...  ...  ...  ...  ...  ...  ...        51 

Pigmentation  of  organs  seen  post  mortem        ...  ...         ...         ...       23 

Mai  de  Caderas  : — 

Disease  of  horses...  ...  ...         ...         ...         ...         ...         ...      113 

Due  to  Trypanosoma  eqiiinum  ...  ...  ...  ...  ...  ...      113 

Geographical  distribution  ...  ...  ...  ...  ...  ...      113 

Malpighian  tubes  in  mosquito  ...  ...  ...  ...  ...  ...     249 

Malta  fever,  leucocyte  variation  in  ...         ...  ...         ...         ...         •••57.59 

Mammalia,    blood-plasma    of    trypanosomes    in.      (See    Trypanosomes, 

mammalian.) 
Mammals,  blood  of,  hremogregarines  found  in  blood  of    ...  ...         ...      105 

Man  : — 

Filaritc  in,  transmission  to  others  and  re-infection  of  individual     ...     258 
Trypanosoma  gambiense  pathogenic  to  ...  ...  ...  ...      113 

Mandibles  of  fleas       ...  ...  ...  ...  ...  ...  ...  ...     285 

Mosquitoes  ...         ...  ...  ...         ...  ...  ...         238,  230 

Mandibulate  mouth,  definition  of     ...  ...  ...  ...  ...  ...      155 

Mansonia         ...         ...         ...         ...         ...         ...         ...         ...         ...     130 

Characters  of        ..  226,228,230 

Egg  of       263 

Pupce  of,  respiratory  tubes  ...  ...  ...  ...  ...  ...      279 

Albipes  carrier  of  Filaria  noctuma      ...         ...         ...         ...         ...     256 

Uniformis  ...  ...  ...  ...  ...  ...  ...     230 

Carrier  of  Filaria  noctuma  ...         ...  ...         ...     256 

Marchflies,  characters  of         ...  ...  ...  ...  ...  ...  ...      167 

Marchi's  method  of  demonstrating  fibrous  degeneration  of  nerve  tissue     320 
Mares  affected  by  dourine       ...  ...  ...  ...  ...  ...  ...      112 

Margaropus,  genus  of  Rhipicephalce  ...         ...         ...         ...         .  .     306 

Mast  cells         ...         ...         ...         ...         ...  ...         ...  ...         ...       61 

Staining  of  ...  ...  ...  ...  ...  ...  ...  69,70 

Mastigophora  (or  Flage/lala)  ...         ...         ...         ...         ...         ...     no 

Found  in  blood    ...         ...  ...         ...  ...         ...         ...         ...       71 

Leishman-Donovan  bodies  assigned  to  ...         ...  ...  ...      122 


INDEX  515 

PAGE 

Maurer's  bodies            ...          ...          ...          ...          ...          ...          ...          ...  69 

Maxillce  of  fleas           ...  285 

Of  mosquitoes      ...  238,239 

May-fly,  characters  of...          ...          ...          ...          ..                        ...          ...  156 

Measurements,  microscopic    ...          ...          ...                       ...          ...          ••■  436 

Representation  of            ...         ...          ...          ...          ...          ■••         •••  437 

Meckel's  diverticulum,  prevalence  among  Chinese             ...          ...          ...  23 

Mediastinum,  posterior,  locality  for  parasites          ...          ...          ...          ...  22 

Megaloblasts  in  the  blood      ...           ...          ...          ...          ...          ...          ...  53 

Megarhina  : — 

Larvoe  of,  respiratory  syphon  in  ...  ■••  ...  •••  ..271 

Megarhinince,  characters  of  . . .          ...          . .           ...          ...          ...          •  ■  •  208 

Eggs  of     ..           262 

Method  of  laying 263 

Mekena  332,333 

Melanin,  absence  of,  not  a  disproof  of  occurrence  of  malaria       ...          ...  313 

Chemistry  of        ...          ...          ...          ...          ...          ...         ...          ■■•  311 

Distribution  of,  in  body...          ...          ...          ...          ...          ...          •■•  312 

Evidence  of  blood  destruction   ...          ...          ...          ...          ...          •••  3l& 

Pigment  deposit  in  malaria        ...  ...  ...  ...  ...   88,311-316 

Melophagns  ovinus      ...         ...         ...         ...          ...          ...         ...         ...  1 99 

Meningiiis,  condition  of  cerebro-spinal  fluid  in      ...                      •■•          •••  481 

Menolepis         232 

Mercury,  perchloride  of,  method  of  fixation  by     ...          ...           ■•          •••  51 

Merozoites        ...          ...          ..           ..           ...          ..                        ...          ...  7° 

Mesentery,  root  of,  locality  for  parasites     ...          ...          ...          ...          •••  22 

Metamorphosis,  definition  of ...           ..          ...           ..          ...          ...         ...  157 

Methseinoglobin  in  "  Blackwater  Fever  "    ...          ...          ...          ...          ...  143 

Spectra  of. ..          ...          ...          ...          ...          ...          ...          ...          ...  143 

Methyl  alcohol  as  solvent  in  staining           ...          ...                      ...          ...  66 

Methylated  spirit,  automatic  Bunsen  burner  for     ...          ...          ...         ...  3 

Methylene  blue  (Griibler's),  solution  of       ...  ...  ...  ...  66,67 

Rendered  polychrome      ...          ...          ...          ...          ...          ...          ...  64 

Treatment  with  oxide  of  silver  ...          ...          ...          ...          ...          ...  64 

Microblasts  in  the  blood         ...         ...          ...         ...          ...           ..         ...  53 

Micrococci         ...         ...         ...         ...         ...         ...         ...         ...         ...  390 

Microfilaria  bancrofti,   location  in   body   when  absent    from    peripheral 

blood 129 

Nuclei  of       128 

Periodicity  of,  alteration  in             ...                       ...          ...          ...  128 

Demarquayi         ...          ...          ...          ...          ...          ...          ...          ...  129 

Diurna,  or  loa     ...          ...          ...          ...          ...          ...          ...          ...  123 

Nuclei  of       128 

Nocttirna,  or  bancrofti    ...         ...         ...         ...         ...         ...         ...  129 

human,  next  stage  of  growth  in  mosquitoes        ...          ...          ...  130 

Ozzardi     ...         ...         ...                   ...         ...         ...         ...         ...  129 

Persians    ...         ...         ..           ...         ...         ...         ...         ...         ...  129 

Microfilaria-,  accurate  depiction  of    ...          ...          ...         ...         ...         ...  128 


5K>  INDEX 

Microfilariae — continued.  PAGE 

In  blood-films       ...          ...          ...          ...  ...  ...          ...  125 

Dried             ...          ...         ...          ...  ...  ...  126 

Characters  of        ...          ...          ...          ...  ...  124,  125 

Examination  of    ...           ..         ...         ...  ...                      ...  125 

Points  important  in...         ...         ...  ...  ...  ..  125 

Human,  developmental  changes  not  effected  in  human  blood  and 

tissues...            ...         ...         ...         ...  ...  ...  ...  ...  129 

Nuclear  core  of,  arrangement    ...          ...  ...  ..          ...  ...  128 

Gaps  in ..          ...  ...  ...  ...  ...  127 

Periodicity  of,  definition             ...          ...  ...  ...  ...  ...  129 

Diurnal          ...         ...          ...         ...  ...  ...  ...  ...  128 

Nocturnal      ...          ...         ...          ...  ...  ...  ...  ...  128 

Size  of       ...          ...         ...          ...          ...  ...  ...  ...  ...  125 

Microgametes  ...          ...         ...          ...         ...  ...  ...  ...  ...  92 

Micrometer  scale  in  microscopic  work          ...  ...  ...  ...  13,  14 

Slide           ...          ...          ...          ...          ...  ...  ..  ...  ...  13 

Micro-organisms  :  — 

Acid-fast    ..           ...          ...          ...          ...  ...  ...  ...  422 

Chief  cultural  characteristics  of  Coli  group  of...  ...  ...  ...  425 

Destruction  by  leucocytes  effected  by  blood-serum  ...  ...  ...  152 

Method  of  enumerating  in  air    ...          ...  ...  ...  ..  ...  447 

In  fluid      ...          ...          ...  ...  ...  ...  ..  447 

In  solids    ...          ...         ...  ...  ...  ..  447 

Non-acid-fast        ...          ...          ...          ...  ...  ...  ...  ...  423 

Retaining  stain  when  treated  by  Gram's  method  ...           ..  422,423 

Not  retaining  stain  when  treated  by  Gram's  method  ...  ...  424 

Proportion  of,  to  blood  count    ...          ...  ...  ...  ...  ...  446 

Substances  formed  in  blood-serum  as  result  of  infection  by  ...  ...  150 

See  also  Organisms 

Microscope,  examination  by,  preparation  of  tissues  for  ...  ...  26 

(Laboratory)         ...         ...          ...          ...  ...  ...  ...  .5,6 

Accessories,  in  use  of         ...          ...  ...  ...  ...  13-20 

Adjustments  for  focussing  objects ...  ...  ...  ...  ...  8 

And  screw  movements            ...  ...  ...  ...  ...  9 

Camera  lucida  for    ...         ...          ...  ...  ...  ...  ...  16 

Condenser                  ...          ...          ...  ...  ...  ...  ...  II 

Correction  of  chromatic  aberration  ...  ...  ...  ...  11 

Cover-glasses  for  objects  under     ...  ...  ...  ...  ...  16 

Definition  of  objects  under             ...  ...  ...  ...  ...  10 

Dissecting     ...         ...         ...          ...  ...  ...  ...  15 

Flatness  of  field  necessary ...          ...  ...  ...  ...  ...  10 

Focussing  of  objects           ...          ...  ...  ...  ...  ...  12 

Illuminating  apparatus        ...          ...  ...  ...  ■■■  ...  8 

Illumination              ...          ...          ...  •••  •••  ■  •■  •■•  II 

Lenses  of      ...         ...         ...          ...  ...  ■••  ...  ...  9 

Cleansing  of      ...          ...          ...  ...  ...  ■  ■•  ...  13 

Deterioration  of            ...          ...  ...  ..  ...  ...  12 

Re-grinding  of...         ...          ...  ...  ...  ••  12 

Testing  of         9 


INDEX 

Microscope  (Laboratory) — continued. 
Magnification 

Micrometer  slide  and  scale  for 
Mirror  for 
Objectives,  testing  of 

Parts  of         

Price  of 

Sub-stage  condenser  for 
Tube  of 

Warm  stages  for 
Microscope  slides 
Microscope  table  for  laboratory 
Microtomes  for  section-cutting 
Midges,  characters  of ... 

(Gall)         

Mimomyia 

Mirror  for  laboratory  microscope 

Mixing  chamber  for  dilution  of  blood-serum 

Monkey,  unnamed  species  of  Piroplasma  found  in,  in  Uganda 

Monkeys,  African  : — 

Plasmodium  kochi in 
Reproduction  of  relapsing  fever  in 
Monocystis  ag  His 

Magna 
Monogenia 

Mononuclear  leucocytes,  staining  of... 
Monostoma  hntis 
Monostomidcs    ... 
Mosquitoes 

Alimentary  canal,  dissection  of... 
Breeding  places  ... 
Carriage  of 
Box  for 
Cage  for 
Carriers  of  Filaria  immitis 

Nocturna,  genera  and  species  comprising 


Characters  of 

External  examination 
Development  of  Filaria  nocturna  in,  demonstration 
Dissection  of 

Freshly-killed  specimens  alone  suitable  for 

Points  to  be  observed  in     ... 
Eggs  of,  examination  of,  points  to  be  observed  in 

Methods  of  laying  in  different  genera 

Retention  of  vitality 
Embedding  of 
Female,  spermathecre  of 
Filaria  in,  demonstration 
Food  for    . . . 


167,  I 


5*7 
PAGE 

II 

I.3-I5 

8 

10 

7 
9 
8 

7 

'5 
16 

2 

34.  37 
.  167 
.  168 
.  232 
8 
.  148 
■  103 

102 
118 
109 
109 

•  354 

55 

•  354 

•  354 

200- 236 
240 

233.  234 
279 

280 
281 
258 
256 
200 

234 
256 
241 
240 
247 
273 
261 
262,  263 

245 
251 
247 
282 


237, 


510  INDEX 

Mosquitoes — continued.  r.v .  i 

Cenital  organs,  female    ..           ...         ...          ...          ...  251 

Male      ...     251 

Ilypopharynx  of  ...          ...          ...          ...          ...          ...          ...  238,239 

Internal  anatomy...          ...          ...          ...          ...          ...          ...  ...     248 

Intestinal  tubes  of,  contain  bacilli  in  large  numbers  ...          ...  ...     259 

Labium  of,  Dutton's  membrane  in        ...          ...          ...          ...  ...     258 

Labrum-epipharynx  of    ...         ...          ...          ...         ...         ...  237,  239 

Larva.-  of,  alimentary  system      ...          ...          ...         ...          ...  ...     271 

Anatomy  of  ...          ...         ...          ...         ..           ...          ...  ...     268 

Appendages  on  eighth  and  ninth  abdominal  segments  ...  ...     273 

Breeding  of,  oxygenation  of  water  for      ...                      ...  ...     274 

Places    ...         ...         ...          ...                      ..           ...  274,  275 

Character  and  peculiarities  attaching  to           ...  ...     267 

Artificial 264,  265,  277,  278 

Natural      266 

"  Brushes "  on  head  ...         ...  ...         ...  ..."       ...     269 

Colouring  of...          ...          ...          ...          ...          ...          ...  ...     274 

Duration  of  stage     ...          ...         ...          ...         ...         ...  ...     274 

Examination  of,  points  to  be  observed  in             ...          ...  ...     273 

Hairs  on  abdomen  in          ...          ...         ...         ...          ...  ...     271 

Head  270 

How  to  obtain          ...         ...          ...          ...          ...          ...  264,266 

Inferior  lip  plate       ...          ...          ...          ...          ...          ...  ...     269 

Intestinal  system      ...         ...         ...          ...          ...         ...  ...     271 

Natural  enemies  of  ...          ...          ...          ...          ...         ...  ...     274 

Respiratory  syphon  ...  ...  ...  ...    233.  269,  271,  273 

Respiratory  system  ...          ...                       ...          ...          ...  272,273 

Tubes  in            ...         ...         ...           ..          ...          ..  268,  269 

Thorax           ...          ...          ...          ...          ...          ...          ...  ...     270 

Transmission  down  stream  by  floating  water-plants       ...  ...     275 

Variation  of  parts  in  different  species       ...          ...          ..  269-271 

Larval  stage,  duration  of           ...          ...          ...          ...          ...  ...     268 

Malpighian  lubes  of        ...          ...         ...          ...          ...         ...  ...     249 

Mandibles  of        .           238,239 

Maxillae  of            238,239 

Metamorphosis  of            ...         ...          ...          ...         ...          ...  157,  158 

Method  of  escape  of  filariae  from           ...         ...          ...          ...  ...     258 

Microscopical  examination  of   ...          ...          ...          ...  ...     201 

Mounting  of        ...         ...         ...         ...         ...         ...         ...  ...     234 

By  Bentley-Taylor  method            235,236 

In  glycerine  jelly     ...          ...         ...          ...         ...          ...  ...     235 

Next  stage  of  growth  of  human  Microfilaria  nocturna  effected  in   ...      130 

(Esophagus,  diverticula  at  commencement      ...         ...          ...  ...     247 

Parasites  in,  development          ...         ...         ...         ...          ...  ...     252 

Position  where  sporozoites  are  found  in  body  of        ...  ...     256 

Protozoa  found  in            ...          ...          ...         ...         ...          ...  ...     259 

Pupse  of 267,272,278,279 

Hatching  out            ...         ...         ...         ...         ...         ...  •■•     279 

Respiratory  tubes     ...         ...         ...         ...         ...         ...  ...     270 


INDEX 

Mosquitoes — continued. 

Salivary  glands,  dissection 

Mounting  of... 
Sections  of,  cutting  and  staining 

Mounting 
Species  of,  identification 
Stomach  of 
Dissection 
Examinaiion 
Mounting 
Mosquito-phase  of  malaria  parasite  ... 
Moth,  characters  of     ... 
Moulds  ... 

Prevention  of  growth  of 
Mucor    ... 
Mucus  :  — 

Examination  of,  in  fceces 

Presence  of  blood 
Mliller's  fluid  :  — 

For  fixation  and  hardening  of  tissues  ... 
Of  tissues 
Mus  decumanus,  see  Rat. 
Muscida : — 

Characters  of 
Larvae  of  ... 
Muscids,  characters  of 
Museum  preparations : — 

Of  organs  and  viscera,  method  of  preserving 
Preservation 

Kaiserling's  method    ... 
Myelocytes : — 

Distinguished  from  eosinophile  leucocytes 
Granules  present  in 
Mononuclear,  resembling  leucocytes 
Staining  of 
Myiasis : — 

Cause  of    ... 
Cutaneous 
Internal 
Alyriapoda 
Myxosporidia    ... 
Myzoniyia  funesta 

Not  a  carrier  of  Filaria  noclurna. 
Rossi,  as  carrier  of  malaria 
Myzorhynchus  barbiroslris,  carrier  of  Filaria  noclurna 
Sinensis,  carrier  of  Filaria  nocluma 


519 
PAGE 

243.  244 
...       250 

246,  247 
...  247 
...       234 

247,  249 
...  247 
...  243 
...       250 

...  77 
...  157 
390,  417 

•  ••  39 
...     420 

331,  332 
■■■     332 

...  28 
...       28 


■  165 

•  158 
167 

25,  26 
24.  25 

■  25 

60,  61 

70 

.       60 

60,  70 

160 
. .  160 
..     160 

•  155 

•  324 

■  253 
257 

,     471 

•  256 
.     256 


Nagana  (or  Tsetse  disease),  disease  resembling,  produced  in  horse  by 

Trypanosoma  dimorphon         ...  ..  ...  ...  ...  ...      1 1 4 

Due  to  T?ypanosorna  brucei      ...  ...  ...  ...  ...  ■■•      112 


520 


INDEX 


Nape,  definition 
Necator  americanus 

Eggs  of 

Negri  bodies    ... 

Negro,  deeply  fissured  lungs  ol 

Weight  of  brain  in,  age  at  which  maximum  i.- 
Nematocera  auoma/a,  characters  of  ... 

Vera,  characters  of 
Nematoda  found  in  blood 

See  also  Nemocera. 
Nematodes 

In  blood  in  lower  animals 

Examination  in  alcohol  and  glycerine  ... 
Treatment  for 

Large,  preservation  and  examination  of 

Permanent  specimens  of 

Small,  preservation  and  examination  of 

Preservation  in  alcohol  ... 
Nemocera  : — 

Characters  of 

Classi6ed  ... 

Auomala,  description  of  families  of 
Neosporidia 

Nessler's  solution,  method  of  preparation   ... 
Nenroptera  : — 

Characters  of 

Metamorphosis  of 
Nitric  acid,  test  for  indican  in  urine 
Nitrites  in  water,  test  for,  qualitative 

Quantitative 
Nitrogen  in  fseces,  determination  of  amount  of 
Normoblasts  in  blood... 
"  No-see-um  "... 
Nutrient  agar   ... 
Nutrient  broth  : — 

Method  of  preparation    ... 

Neutralization  of... 

Solid  media 

Sterilization  of     ... 
Nutrient  gelatine 

Nuttall,  on  Anopheles  maculipennis 
Nycteribida,  characters  of 
Nymph,  definition  of  ... 
Nyssorhynchus,  characters  of 

Fuliginosus 

Occirur,  definition  of 
(Esophagus  of  mosquitoes 
Oil  immersion,  use  of... 


PACB 

'63 

360 

343-  344 

32  5.  326 

23 

is  attained 

22 

163,  167 

167 

71 

345357 

124 

134,  135 

'34 

47S 

357 

477.  47S 

134 

161 

166 

172 

324 

43o 

156 

157,  158 

374 

431.432 

43I.432 

338 

53 

171 

$84 

378 

379 

383 

382 

383 

278 

199 

'58 

225 

225 

162 

247 

53 

INDEX 

Oliver's  tintometer 

Oncospheres  (eggs  of  tape- worm) 

Oocysts  of  coccidia 

Ookinet... 

Definition  of 
Opisthorchis 
Opsonic  index  :  — 

Definition  of 

Method  of  obtaining 
Opsonins 

Definition  of 
Organisms  (aerobic)    ... 

(Anaerobic) 

Chemical  products  of 

Conditions  affecting  growth  of  ... 

Differentiation  by  method  of  staining 

Flagella  on 

Growth  on  artificial  and  solid  media    ... 

Koch's  postulates  as  to  pathogenicity  ... 

Method  of  demonstrating  capsules  on  ... 

Serum  reactions  of 
Organs  : — 

Abnormalities  of,  peculiar  to  various  races 

Average  weights  of,  in  Europeans,  Negroes,  Indians 

Method  of  preparing  museum  specimens  of    ... 
Oriental  sores  ... 
Ornithodorus : — 

Moubata,  caused  by  ticks 

Description  and  geographical  distribution 

Savignyi,  description  and  geographical  distribution 
Orthoptera : — 

Adult  development  of     ... 

Description 
Orthorrhapha : — 

Classification  of    ... 

Brae hycera,  description  of  families 
Orth's  fluid,  fixation  of  tissues  by 
Osmic  acid  mixtures,  fixation  of  tissues  by  ... 
Owl  :— 

(Little),  blood  of,  Trypanosoma  nocture  found  in 

Midges,  character  of 
Oxyhemoglobin,  spectra  of    ... 
Oxyuris  vermicularis  : — 

Characteristics  of. .. 

Kggs of      


and 


52» 

PAGE 

...      448 

342,  343 

322-  323 

93-  253 

■••       93 

■••     354 

•■•  153 
'52,  153 

...  152 
...  152 
■••  403 
■  •  ■  403 
. . .  406 
...  403 
■■•  395 
•■  393 
400,  401 
...     411 

•••     393 

...     407 

22,  23 

Chinese  ...     477 

25,  26 

328 


}o8 


306 
307,  308 

...     158 
...     156 

166,  167 

...     174 


102 
171 
J43 


342 


357 
343 


Pangonia,  characters  of 

Paraffin,  English,  unsuitable  for  the  Tropics 


^75 
52 


> 


INDEX 


Para  (tin  —continued. 

Kmbedding  method 

Modification  of 
Rapid 
Mixtures  for  the  Tropics 
Removal  of,  from  sections 
Sections,  culling  of 

Fixation  of,  on  slide 
Paragonimus    ... 
Westcrmani 
ParatnphiitomidiC 

Parasites  allied  to  those  of  human  milaria  found  i 
(Animal),  found  in  blood 

Staining  of,  in  tissues 
Blood-corpuscles  containing 
Found  in  blood-plasma  ... 
In  blood,  staining  of 

Of  animals    ... 
Embryos  passed  in  faeces 
In  faeces,  method  of  straining  out  of  fceces 
Microscopic  examination    ... 
Species  passed  naturally  and  after  anlhel 
Varieties  and  characteristics  of  ova 
In  tissues  ... 

Helminths    ... 
Protozoal 
IL'jmatozoal,  carried  by  ticks    ... 
Intestinal  ... 
Measurements  of  ova 
(Non-protozoal),  in  human  blood 
Root  of  mesentery  and  posterior  mediastinum 
Vacuolated  coipuscles  mistaken  for 
Parasitism,  internal 

Patton,  description  of  mammalian  hcemogregarine 
Pediculi,  characters  of 

Geographical  distribution,  variation  under 
Pediculidce 

Characters  of 
Pediculus 

Capitis  (head  louse) 
Vestiiiienli  (body  louse)  ... 
Pedipalpi  (whip  scorpions) 
Pellagra,  caused  by  Aspergillus  fumigatus 

Leucocyte,  variation  in   ... 
Penicillium 

Pentastomuin  conslriclum 
Perchloride  of  mercury,  method  of  fixation  by 
Pettenkofer's  reaction  for  detection  of  bile  acids  in  faeces 
Phenol  sulphonic  acid,  method  of  preparing 


30 

32 

33 

32 

...       39 

...       37 

38 

...     356 

•••     350 

352,  356 

i  animals 

102 

7i 

...       28 

•40,  41 

...     no 

...       62 

100 

...     344 

339,  340 

34°-344 

mintics 

...     344 

34'-344 

321-329 

328|  329 

321-328 

•••     303 

•••     345 

-     344 

...      124 

,  locality  for  . 

22 

45.  46 

...     159 

...      106 

...      157 

...     293 

...     291 

...     291 

...     292 

...     292 

...     292 

...     296 

...     420 

...       59 

...     420 

...     309 

...       51 

i  faeces 

•     333 

...     432 

INDEX 


52S 

I'AGE 
■   232 
171 
232 

...   292 

293 

.   2SO 

3I5.3I6 
3II-32O 

...       316 

3*4.  315 

313-316 


Philodendromyia 
Phlcbotomus 
Phoniomyia 
Phlhirius 

lnguinalis  (crab  louse)    ... 
Picrocarmine  for  staining  malaria  parasites 
Pigment  deposits,  accompanied  by  urobilin  discharge 
And  degeneration  in  tissues   ... 
In  skin  ... 

Staining  for  examination  of    ... 
Yellow  ...  ...  ...  ...  ...  .... 

Piroplasma  : — 

Carried  by  ticks  ... 
Development  of  ... 

Asexual  cycle   ...  ...  ...  ...       .... 

Sexual  stage     ... 
In  blood-plasma 
Species  unnamed  found  in  monkey  in  Uganda 
Bigemimtm  cause  of  Texas  fever  of  cattle 

Developmental  forms  in  Rhipicephalus  ausiralis,  R.  everlsi, 
and  Hyalonwia  cegypticum  ... 
Transmission  of,  in  America,  Africa  and  Queensland  ... 
And  P.  parvwn  harboured  by  one  animal  at  same  time 
Cams  cause  of  epidemic  jaundice  in  dogs 

Cannot  reproduce  disease  in  other  animals 
Developmental  forms  of,  in  Rhipicephalus  sanguineus  ... 
Insects  transmitting,  in  South  Africa,  Europe  and  India 
Equi 

Produces  disease  in  horses  and  donkeys  only     ... 
Muris 
Ovis 

Carrier^  of    ... 
Parvum,  cause  of  Rhodesian  fever  in  cattle   ... 
Piroplasmala    ... 

Difference  of,  from  Hcemamcebcp  ...         ...         ...  .  •  102 

Intermediate  hosts  of      ...  ...  ...  ...  ...  .  ••■      103 

Piroplasmoses,  caused  by  ticks         ...  ...  ...         ...         ...         3°3>  3°4 

Pityriasis  versicolor     ...  ..  ...  ...  ...  ...  ..  •••     4X7 

Plague,  rats  hosts  of  bacillus  of         ...  ...  .  ...  ...  •■•      283 

Plasmodium  kochi  in  African  monkeys         ...  ...  ...  ...  ...      102 

Platelets  (see  Blood  platelets). 

Plating  on  agar  ...  ...  ...  ...  ...  ...  ...  ...      387 

Plehn's  bodies ...  ...  ...  ...  ...  ...  ...  ...  ...       69 

Pleura,  definition  of    ...  ...  ...  ...  ...  ...  ...  ...      163 

Pneumonia,  leucocyte  variation  in     ...  ...  ...  ...  ...  58,  59 

Pneumono-mycosis,  caused  by  Aspergillus  ...  ...  ...  ...     420 

Poisson's  formula         ...  ...  ...  ...  ..  ...  ••         451,452 

Applied  to  experiments  in  malaria  carried  out  in  Central  Africa     ...     473 
Carried  out  in  Klang      ...         ...  ...  ...  ...     472 


303 

104 
104 
104 
no 
103 
103 

104 
103 
103 

103 
103 
104 
103 
103 
103 
103 

103 
103 

i°3 

102 
io^ 


5-M 


INDEX 


232 
56 

277 

454 

364 

3^9 

21-39 

23 

24 

21 

24 
23 
21 


PolyUpidomyia 

Polymorphonuclear  leucocytes,  staining  of... 

Pools  on  shores  of  lakes  as  breeding  places  for  mosquito  larvae 

Population,  statistics  dealing  with  malaria  as  regards 

Pork,  infection  of  man  with  Trichina  spiralis  through    ... 

Porocephalus  armillatus 

Postmortem  examinations  in  Tropics 

Abnormal  appearances  mistaken  for  disease   .. 

Emphysematous  distension  of  organs  seen  at... 
Differences  from  those  in  temperate  climates... 
Examination  for  entozoa  ...  ...  .  . 

Putrefaction  of  organs     ... 

Removal  of  thoracic  and  abdominal  viscera  en  masse 
Potassium  acetate,  glycerine  and  water,  preservation  of  museum  prepara- 
tions in...         ...         ...          ...         ...         ...          ...  25.  26 

Nitrate,  method  of  preparing  standard  solution  of  ...     432 

Precipitins        ...          ...          ...          ...         ...          ...  .150 

How  obtained      ...          ...          ...         ...          ...  150,  151 

Prosolepis  232 

Proteosoma  of  birds 100,200 

Host  of 229 

Protozoa           ...         ...          ...          ...          ...          ...          ...  364 

Examination  of  blood  for           ...          ...          ...  71 

Found  in  mosquitoes       ...          ...          ...          ...          ■■■  259 

Pseuttograbkamia        ...                     ...                     ...  232 

Pseudoscorpionida:  (book  scorpions)               ...          ...  ...                  296 

Pscudouranotcenia       ...          ...          ...          ...          ..           ...  231 

Psilosis  ...          ...          ...          ...          ...          ...          ...          ...  ...                 337 

See  also  Sprue. 

Psorophora,  egg  of       ...          ...          ...          ...  263 

Salivary  glands  of            ...          ...          ...          ...          ■■•  250 

Psychodida,  characters  of       ...          ...          ...          ...          . . .  1 7 ' 

Ptilinum,  definition  of            ...         ...         ...         ...  163 

Pitlex 289 

Larva  of   ...          ...          ...          ...          ...         ...          ■  ■•  2^S 

Cheopsis 288,  289 

Geographical  distribution  ...          ...  ...     289 

Irritans     ...          ...          ...          ...          ...          ...          ...  •■■      28S 

Pulicida           •••     287 

Characters  of       287,289 

Feeding  time                    ...           ..                      ..  ...      150 

Genera  of...          ...          ...          ...  •••     289 

Pulvilli,  definition  of ...      165 

"Punkies"      ...  171 

Pupa  and  metamorphosis       ...          ...          ...  ...      158 

Pupae  of  mosquitoes 267,272,278,279 

Pupipara,  characters  of          161,  167,  197 

Putrefaction  of  organs  seen  in  post-mortem  examinations  in  Tropics        ..        23 

Pyrelofliortis  costalis,  carrier  of  Filaria  nerturna               ...  ...      256 


INDEX  525 

PAGE 

Queensland,  transmission  of  Piroplasma  bigemimim  in  ...  ...      103 

Quinine,  excreted  in  urine,  estimation  of    ...          ...          ...  ...  ...     377 

Rabies,  in  do^s,  occurrence  of  negri  bodies           ...          ...  ...  ...     325 

Races,  yarious  bodily  abnormalities  peculiar  to     ...          ..  ...  ...        23 

Rat  (A/us  decumanus),  hcemogregarine  leucocytes  of        ...  ...  106,  107 

Rats,  reproduction  of  relapsing  fever  in       ...          ...          ...  ...  ...      118 

Full-grown,  species  of  try panosomes  non-pathogenic  to  ...  ...      112 

Healthy,  infection  with  Trypanosoma  lewisi               ...  ...  ...      283 

Host  of  Bacillus  pestis  283 

Razor,  position  of,  in  microtomes     ...         ...         ...          ...  ...  36,  37 

Reagents,  mounting  and  embedding,  used  in  tropical  work  ...  ...     482 

Used  in  tropical  laboratory  work          ...          ...          ...  ...  ...     482 

Recurrent  fever,  spirochaeta  producing         ...          ...          ...  ...  ...      117 

Reduviidce                                ...         ...         ...          ...         ...  •■•  ...     295 

Predatory  qualities  of  some  members,  useful                ...  ...  .  .     295 

Relapsing  fever,  animals  to  which  pathogenic        ...          ...  ...  ...      118 

Leucocyte  variation  in                ...          ...          ...          ...  •■■  ...       59 

Enlargement  of  spleen  in           ...          ...          ...          ...  ...  ...      119 

Symptoms              ...          ...          ...          ...          ■•■          ■••  ■■•  118,  119 

Respiratory  syphon,  in  larvae  of  mosquitoes  ...  ...   233,  269,  271,  273 

Respiratory  tubes,  in  mosquito-larvae           ...          ...          ...  ...  268,269 

Of  mosquito-pupae                         ...          ...          ••■          ■■•  ■  ...     279 

Rhinosporidium  kinealyi       ...         ...         ...          ...          ■•■  ••■  325,326 

Rhipicentor       ...          ...          ...          ...          ...          •■■           ••  •••  ...      306 

Rhipicephalae    ...          ...          ...          .  .          ••■         •••          ■•■  ■■■  305,  306 

Rhipicephalus  ...          ...          ...          ...          ...          ...          ...  ...  ...     306 

Rhipicephalus   annttlalus,    transmission  of  Piroplasma   bigemimim    in 

America  by       ...          ...          ...          ...          ...          ...  ...  ...      103 

Australis,  developmental  forms  of  Piroplasma  bigemimim  in  ...     104 

Bursa,  carrier  of  Piroplasma  ovis         ...          ...          ...  ...  ..       103 

Evertsi,  developmental  forms  of  Piroplasma  bigemimim  in...  ...      104 

Sanguineus,  carrier  of  Piroplasma  canis,  in  India     ...  ...  ...      103 

developmental  forms  of  Piroplasma  cam's  in       ...  ...  ...      104 

Transmission    of  Piroplasma   bigemijium   in    Africa   and    Queens- 
land by               ...          ...          ...          ...          ...          ...  ...  ...      103 

Rhodesian  fever  of  cattle,  parasite  causing              ...          ...  ...  ...      103 

Rhytichopsylla  ...          ...          ...          ...          ...          ...          ...  ...  ...      290 

Rhyncotce,  see  Hemiptera. 

Rivers,  as  breeding-places  for  mosquito-larvte        ...          ...  ...  ...      276 

Robber-flies,  characters :of     ...          ...          ...          ...          ...  ...  ...      167 

Rogers,  L.,  nature  of  Leishman-Donovan  bodies  ...          ...  ...  ...      122 

Romanowsky  : — 

Method  of  staining           ...          ...          ...          ...          ...  ■  ...  61,79 

For  chromatin,  modifications         ..           ...          ...  ...  66 

See  also  Leishman's  modification. 

Ross,  R.,  F.R.S.,  on  leucocyte  variation     ...          ...          ..  ...  ...       57 

Ross's  method  of  measuring  blood   ...          ...          ...         ...  ...  ...     445 

Round-worm,  eggs  of              ...         ...         ...          ...         ...  ..  ...     341 


20 


INDEX 


Sabethes       

Characters  of 
Sabcthinits  sabethoides 
Sacchaiomycetes 
Saline  solution,  isotonic  strength  of 

Estimation    ... 
Salivary  glands  of  mosquitoes 

Dissection 
Salt  and  ice  freezing  mixture 
Sambon,  L.  W.,  box  designed  by,  for  carriage  of  mosquitoes 
Sand-flies,  character  of 
Sarcocystis 
Sarcopsylla 

Penetrans  (Jigger  or  Chigoe) 
Characters  of 

Geographical  distribution  ... 
Parasitic  in  man 
Sarcopsyllidie,  characters  of  . . . 
Sarcopudse 
Sarcospoiidia    ... 

"  Rainey's  capsules,"  or  "  Aliescher's  tubes  " 

Staining  for,  demonstration  of  ... 
Scale-insects,  characters  of    ... 
Schaudinn  :  — 

Life-cycle  of  Trypanosoma  nortua 

Relationship  of  Spirochetes  to  Trypanosomas 
Schistosoma  :  — 

Eggs  of      

Geographical  distribution  of 
Schistosomidcc  ... 
Schistosoiinim  hcematobmm 

Characteristics  of  eggs     ... 

And  japoniaim    ... 

Japonicuvi 
Eggs  of 
Schizogregarimc 
Schizomycetes    ... 

Morphology  of     ... 

Motiliiy  of 

Spore  formation  of 

Structure  of 
Schizonts 

Schizophora,  characters  of 
Schizotrypanum  (see  Trypanosoma  cruzi.) 
Schmidt's  reaction 
Schuffner's  dots 
Scorpions,  characters  of 
Scorpionida       ...    • 


'44. 


243- 


232 
168 
232 
421 
'44 
'45 
250 
244 

35 
280 

172 

325 

290 

160,  288,  290 

290 

291 

290 

290 

297 

324-326 

324 

324.325 
157 

102 

120 

372 

371 

356 
...71,  124,  350 

343 

328,  329 
...71,  124,  350 

344 

108 

390 

390 

••  391 

392 

393 

76 

167 

333 

68,  69.  90,  105 

155 

296 


INDEX  527 

PAGE 

Screw-worm      ...          ...          ...          ...          ...  ...  ...  ...  160 

Fly,  geographical  distribution  ...         ...         ...  ...  ...  ...  194 

Scurvy,  increase  of  lymphocytes  in   ...          ...          ...  ...  ...  ...  58 

Scutellum,  definition  of          ...          ...          ...         ...  ...  ...  ...  163 

Section-cutting  :  — 

Instruments  for    ...         ...          ...          ...          ...  ...  ...            34,  37 

Methods  of            ...          ...          ...          ...          ...  ...  ...             35,  36 

Sections  : — 

Celloidin,  cutting  of        ...          ...          ...          ...  ...  ...  ...  38 

Paraffin,  cutting  of          ...          ...          ...          ...  ...  ...  ...  37 

Removal  of  paraffin  from             ...          ...          ...  ...  ...  ...  39 

of  xylol  from             ...          ...          ...          ...  ...  ...  ...  39 

Treatment  of,  after  cutting        ...          ...          ...  ...  ...  ...  39 

Sepsis,  leucocyte  variation  in             ...         ...         ...  ...  ...             38,59 

Serum  reactions  of  organisms             ...          ...          ...  ...  ...  ...  407 

Sexual  organs,  development  of,  in  insects   ...          ...  ...  ...  ...  158 

Sheep,  Piroplasma  ovis  found  in       ...          ...          ...  ...  ...  103 

Sheep  ked         ...         ...         ...         ...         ...         ...  ...  ...  ...  199 

Shelves  for  laboratory              ...          ...          ...          ...  ...  ...  2 

Silver,  oxide  of,  treatment  of  methylene  blue  with  ...  ...  ...  64 

Simpson,  G.  C.  E.,  on  urobilin  in  malaria  and  extraction  from  faeces  333,  334 

Simulirire,  characters  of          ...          ...          ...          ...  ...  ...  ...  167 

Siphonaptera     ,.           ...          ..           ..           ...          ...  ...           ..  ...  283 

Characters  of        ...          ...          ...          ...          ..  ...  ...  icy 

Siphimciilaia    ...          ...          ...          ...          ...          ...  ..  ...  ...  291 

See  also  Anopleura. 
Skin : — 

Pigment  deposits  in          ...          ...          ...          ...  ...  ...  ...  316 

Tropical  fungi  attacking              ...          ...          ...  ...  ..  ...  417 

Skin  organisms,  contamination  of  blood  films  with,  during  preparation...  139 

Skusea...           ...         ...         ...         ...         ...          ...  ...  ...  ...  231 

Slate,  slab  of,  good  background  for  searching  tissues  for  filaria.-  ...  ...  131 

Sleeping  sickness,  due  to  Trypanosoma gambiense  ...  ...  ...  1 1 } 

Late  stage  of  trypanosomiasis    ...          ...          ...  ...  ...  ...  113 

Slides  for  blood  examination,   preparation  of         ...  ...  ...  ...  42 

Method  of  preparing  blood  films  on     ...          ...  ...  ...  ...  48 

Preparation  of  dried  films  by    ...          ...          ...  ...  ...              48.49 

Smegma  bacilli             ...          ...          ...          ...          ...  ...  ...  ...  400 

Snakes,  pigmented  parasite  in  {Hcemocystidiuni)    ...  ...  ...  ...  102 

Snegg I74 

Snipe-flies,  characters  of        ...         ...          ...          ...  ...  ...  ..  ifrj 

Sodium,  citrate  of,  mixture  with  blood  to  prevent  coagulation 141 

Soldier-flies,  characters  of     ...          ...          ...          ...  ...  167 

Sparganum       ...          ...          ...          ...          ...          ...  ...  ...  ...  345 

Spectra  : — 

Of  haemoglobin,  reduced              ..          ...          ...  ...  145 

Of  niethsemoglobin          ...          ...         ...         ...  ...  ...  ...  xa-> 

Of  oxyhaemoglobin           ...          ...          ...          ...  ..,  ...  ...  j.^ 

Spectrum,  manipulation  in  spectroscopic  examination  of  blood 143 


528  INDEX 

PAGE 

Spermathecae  of  female  mosquito      ...          ...          ...          ...  ...     251 

Spinal  puncture,  method  of  performance                 ...  .481 

Spirilla...          ..           ...          ...          ...          ...          ...          ...  390 

Spirit,  second  bath  of,  for  museum  preparations   ...  25,  26 

Spirochxta        ...         ...          ...          ...          ...          ...          ...  117 

Carried  by  ticks  ...          ...          ...          ...          ...          ...  ...  303 

Present  in  yaws   ...          ...         ...          ...         ...         ...  ...  ...     119 

Spirochctta  duttoni : — 

Cause  of  African  tick  fever        ...         ...          ...  ...     118 

Easily  inoculated  into  lower  animals   ...  ...  ...     118 

Spirochctta  pallida  : — 

Demonstration  by  Indian  ink  method...          ...          ...  ...  ...      120 

Spirochxta pertenuis,  present  in  yaws          ...          ...          ...  ...  ...      119 

Recurrentis,  cause  of  relapsing  fever  ...          ...          ...  ...  ...      117 

Morphology  of         ...          ...                       ..           ...  ...  ...      117 

Staining  of 117,  118 

Spirochaetae       ..           ...         ...         ...         ...         ...         ...  ...  ...       71 

Relationship  to  trypanosomes   ...         ...          ...         ...  ...  ...      120 

Spirochaetes,  Levaditi's  method  of  staining           ...          ...  ...  479,  480 

Spleen,  abnormal  size  of,  in  races  indigenous  to  Tropics  ...  ..  ...       23 

Enlargement  of,  in  relapsing  fever       ...         ...          ...  ...  ...      119 

Leishman-Donovan  bodies  present  in  large  numbers  in  ...  ...      120 

Sporoblasts       ...          ...          ...          ...                       ...          ...  ...  76,  254 

Sporogony        ...         ...         ...          ...         ..           ...         ...  ...  ...       77 

Sporozoa,  found  in  blood      ...         ...          ...          ...         ...  ...  ...       71 

Sporozoites,  demonstration  of           ...         ..           ...          ...  ...  255,256 

Formation  of        ...         ...          ...                      ...          ...  ...  ...     254 

Position  where  found  in  body  of  mosquito      ...         ...  ...  ...     256 

Springs  as  breeding-places  for  mosquito  larvae       ...          ...  ...  ...     276 

Sprue 335 

Examination  of  fasces  in...         ...          ...          ...          ..  ...  337,339 

Squa?nomyia     ...          ...          ...          ...          ...          ...          ...  ...  ...      232 

Squirrel    (Kathiawar)  {Funambuhts  petinantii),  hsemogregarine  parasite 

in            106,  107 

Staining  by  Gram's  method  ..                                   ...          ...  ...  ...     395 

By  haematoxylin  ...         ...         ...          ...          ...          ...  ...  ...       52 

By  Romanowsky  method            ..:          ...          ...          ...  ...  ...       61 

Leishman's  modification     ...          ...       63.  64,  75,  78,  80,  94,  95,  123 

For  amyloid  degeneration          ...          ...          ...          ...  ...  ...     319 

For  demonstration  of,  coccidia...          ...          ...          ...  323 

Eggs  and  larvae  of  helminths         ...                      ...  329 

Leishman-Donovan  bodies             ...          ...          ...  ...  327,  328 

For  examination  of  pigment  deposits   ...          ...          ...  314,  315 

For  fatty  degeneration    ...          ...          ...          ...          ...  ...  3 ' 7"3T9 

For  fibrous  degeneration  of  nerve  tissue          ...          ...  ...     319 

Of  blood  platelets            ...          ...          ...          ...          ...  ...  ...        53 

Of  dried  films       ...          ...          ...          ...          ...          ...  5 1-54 

Of  fresh  films,  methods  of          ...          ...         ...          ...  ...  ...       47 

Of  leucocytes        ...          ...         ...         ...          ...          ...  ...       54 


INDEX 

Staining — continued. 
Of  myelocytes 

Of  red  corpuscles... 

Of  white  corpuscles 
Staining-methods,  differentiation  of  organisms  by... 

Enumeration  of  ... 
Stains,  double  ... 

Flushing  off  of     ...  ...  ... 

Simple 

Ziehl-Neelson's  method 
Stallions,  affected  by  dourine 
Staphylococci    ... 
Statistics:  — 

For  tropical  work 

Method  of  deriving 

Method  of  indicating  results  of 

Necessity  for  correction  in 

Value  of  evidence  concerning    ... 
Stegomyia : — 

Character  of 

Egg-laying  of       

Eggs  of 

Larvre  of,  respiratory  syphon  in 
Hairs  on  abdomen  in 
Stegomyia  calopus  (fasciata)  ... 

Eggs  of,  retention  of  vitality     ... 

Transmission  of  yellow  fever  by 

Scutellaris . . . 
Sterilizer  (hot  air) 

(Steam,  Koch's)  ... 
Stomach  of  mosquitoes 

Dissection  .. 

Examination 
Stomoxys,  character  of 

Calcitrans,  carrier  of  surra  {Trypanosoma  evansi) 
Stools,  see  Fasces. 

Strainer,  for  removing  parasites  from  faeces 
Stream-dams  as  artificial  breeding-places  for  mosquito  larvae 
Streams  as  breeding-places  for  mosquito  larvae 
Streptococci 
Streptothrix 
Slreptothrix  madura  ... 

Geographical  distribution  of 
Slrongy  hides  intestinalis  and  cesophagostoma 

Intestinalis 

Embryos  passed  in  faeces 

See  also  Anguillula  intestinalis. 
StreblidcE 
Strophantkus,  arrow-poison  ... 


529 

PAGE 

60 

53 

54 

395 

479 

,  480 

62 

52 

395 

396 

112 

39' 

45i 

45i 

453 

474: 

.  475 

453 

208, 

,  229 

261 

,  262 

262: 

,  263 

271 

271 

229. 

,  256 

263 

259 

229 

1! 

J,  19 

247, 

249 

241 

243 

167 

112 

339. 

340 

278 

276 

39o 

391 

412 

412 

328 

36i 

34i, 

344 

199 

482 

53o 


INDEX 


Strychnos  tiente,  arrow-poison 
Submucosa,  Leishman-Donovan  bodies  in  ... 
Suctorial  mouth,  definition    ... 
Surra,  cattle  often  recover  from 

Fatal  to  horses     ... 

Geographical  distribution 

Trypanosome  of  ... 
Swamps  as  breeding-places  for  mosquito  larvae 
Swift's  freezing  microtome     ... 
Syringe,  hypodermic,  cultivation  of  organisms  from  blood  drawn  from 

vein  by  ... 
Syrphui  flies    ... 


PAGE 

482 
120 

•55 
112 
1 12 
1 12 
112 
277 
35 

140 
167 


Tabanida  : — 

Characters  of 

Feeding  time 
Tabanus,  characters  of 
Tables  suitable  for  laboratories 
Tcznia  echinococcus  : — 

Adult  stage  of      

Definitive  host  of... 

Embryonic  form  ... 
Tachinidce,  characters  of 
Tallquist's  haemoglobin  scales 
Tapeworm : — 

Derives  its  nutriment  by  osmosis 

Eggs  or  oncospheres  of 

Genital  pores  in  Proglottides     ... 

General  structure... 

Man  definitive  host  of     ... 

Method  for  permanent  specimens 

Organs  of  generation,  male  and  female 

Points  to  observe  in  examination 
Tapeworms  (canine),  characteristics  of 
Teeth,  age  for  cutting 
Telosporidia 
Termite,  characters  of 
Tetanus,  arrow-poison  causing 
Texas  fever  of  cattle,  parasite  causing 
Tetrads... 

Theobald,  on  mosquitoes  {Culicina) 
Therioplectes,  characters  of  ... 
Thoracic   and    abdominal    viscera,    remova 

examinations 

Methods  for... 
Thoracic  segments  of  fleas    ... 
Thread-worm,  eggs  of 
Thrips,  characters  of  ... 


2  -5 
in  po. 


161,  167,  174 
...  159 
...   175 


350 
•••  345 
••■  345 

197 
...  450 

•  346 
342,  343 
■•■  349 
...  346 
...  346 
■••  347 
•••  347 
•■•  349 
•  35i 
...  456 
...     324 

.  ...  156 
...  481 
...  103 
...     390 

227,  228,  234 
...     175 
t-mortem 

21 

21,  22 

...       285 

342,  343 
...     157 


INDEX  531 

PAGE 

Thrombosis,  process  in  blood-vessels  in  malaria  wrongly  described  as  ...       87 

Thysanoptera,  characters  of  ...          ...          ...  ...  ...  ...  ...      157 

Thy  samcra       ...         ...          ...          ...          ...  ...  ...  ...  ...      156 

Tick  (sheep),  character  of     ...          ...          ...  ...  ...  ...  ...      161 

Tick  fever,  African  spirochoeta  producing    ...  ...  ...  ...  ...      118 

Tick-flies,  characters  of           ...          ...          ...  ...  ...  ...  ...      167 

Ticks  :  — 

As  carriers  of  disease      ...          ...          ...  ...  ...  ...  303,304 

Dissection             ...          ...          ...          ...  ...  ...  ...  ...     302 

Examination         ...          ...          ..           ..  ...  ...  ...  300-302 

Families  of            ...          ...          ...          ...  ...  ...  ...  297-310 

Feeding  on  infected  animal,  not  infective  ...  ...  ...  ...     104 

But  hand  infection  to  offspring     ...  ...  ...  ...  ..;     104 

Internal  anatomy             ...          ...          ...  ...  ...  ...  302,303 

Intermediate  hosts  of  Piroplasmata      ...  ...  ...  ...  ...     103 

Life-history          ...         ...         ...          ...  ...  ...  ...  ...     303 

Systematic  classification  of         ...          ...  ...  ...  ...  304-306 

Tin  in  water  :  — 

Test  for,  qualitative        ...          ...          ...  ...  ...  ...  ...     428 

quantitative      ...         ...          ...  ...  ...  ...  ...     429 

Tinea  imbricata  (tropical  ringworm)             ...  ...  ...  ...  ...     417 

Tipnla,  characters  of  ...          ...         ...           ..  ...  ...  ...  ...     164 

Tissues: — 

Degeneration  in  ...         ...          ...          ...  ...  ...  ...  311-320 

Fixation  and  hardening  of         ...  ...  ...  ...  ...       27 

And  hardening  in  alcohol  ...          .  .  ...  ...  ...  ...       27 

Time  required  for    ...          ...          ...  ...  ...  ...  28,29 

Imbedding  of       ...          ...          ...          .  .  ...  ...  ...  •••30-33 

Parasites  in           ...         ...         ...          ...  ...  ...  ...  321-329 

Preparation  for  microscopic  examination  ...  ...  ...  ...       26 

Preservation  in  bottles    ...          ...          ...  ...  ...  ...  ...       27 

Toisson's  fluid...         ...          ...         ...          ...  ..  ...  ...  ...     150 

Use  of,  in  blood  counts  ...          ...          ...  ..  ...  ...  ...     438 

Torulce  ...          ...          ...          ...          ...         ...  ...  ...  ...  ...     421 

Toxins  ...          ...         ...         ...          ...          ...  ...  ...  ...  ...      150 

Travers,  report  by,  as  regards  prophylaxis  in  malaria  ...  ...  470,  471 

Trematodes      ...          ...          ...          ...          ...  ...  ...  ...  ...     345 

Eggs  of 343 

Found  in  blood    ...          ...          ...          ...  ...  ...  ...  ...       71 

Large,  preservation  and  examination  of  ...  ...  ...     478 

Small,  preservation  and  examination  of  ...  ...  ...  ...     47S 

Where  found  in  man       ...          ...          ...  ...  ...  ...  ...     350 

Trichina  spiralis : — 

Found  in  intestine  of  man,  pigs,  &c.    ...  ...  ...  ...  ...     362 

Man  infected  by,  from  pork       ...          ...  ...  ...  ...  ...     364 

Method  of  examination  for         ...          ...  ...  ...  ...  ...     362 

Structure  of  male  and  female     ...          ...  ...  ...  ...  ...     362 

Trickinella  spiralis     ...          ...          ...          ...  ...  ...  ...  328,329 

Trichinosis,  increase  of  ecsinophiles  in        ...  ...  ...  ...  ...       58 


;32 


INDEX 


PAGB 

Tricliocephaius  dispar. ..          ...          ...          ...          ...          ...  ...  ...  358 

Characteristics  of  eggs  of  ...         ...  ...         ...  ...  311,342 

Trichomonas,  characteristics  of         ...          ...          ...          ...  ...  ...  367 

Trombididce     ...         ...         ...         ...         ...          ...          ...  ...  ...  297 

Trypanosoma  brucei  causing  nagana  or  tsetse-fly  disease 112 

animals,  wild  or  domesticated,   to  which  pathogenic  ...  ...  112 

Cruzi        in 

Characteristics  of    ...         ...         ...         ...          ...  ...  ...  114 

Carrier  of                 ...         ...         ...         ...         ...  ...  114 

Illness  caused  by     ...         ...         ...         ...         ...  ...  ...  114 

Dimorphon,  geographical  distribution...         ...          ...  ...  ...  114 

Mammalian  type     ...         ...         ...          ...         ...  ...  ...  112 

Producing  disease  in  horse  resembling  nagana  ...  ...  ...  114 

Eqiiimim,  causing  mal  de  Caderas      ...         ...         ...  ...  ...  113 

Gambiense,  carrier  of      ...         ...         ...         ...         ...  ...  ...  116 

Cause  of  sleeping  sickness...         ...         ...         ...  ...  ...  113 

Mammalian  type     ...         ...         ...         ...         ...  ...  ...  in 

Pathogenic  to  man  ...          ...         ...         ...         ...  ...  ...  113 

Evansi,  how  distinguished  from  T.  brucei     ...         ...  ...  ...  112 

Surra  caused  by       ...         ...         ...         ...         ...  ...  ...  112 

Lewisi,  infection  of  healthy  rats  with...         ...          ...  ...  ...  283 

Mammalian  type     ...          ...         ...         ...         ...  ...  ...  Ill 

Nanum,  mammalian  type           ...         ...          ...         ...  ...  ...  ill 

Parasitic  in  cattle     ...         ...         ...         ...         ...  ...  ...  113 

Symptoms  produced  by      ...         ...         ...         ...  ...  ...  113 

Noctuce,  found  in  blood  of  little  owl     ...          ...         ...  ...  ...  102 

Life-cycle  of...         ...         ...         ...         ...          ...  ...  ...  102 

Rhodesiense,  cause  of  trypanosomiasis  of  obstinate  type  ...  117 

Theileri,  cattle  alone  susceptible  to      ...         ...         ...  ...  ...  113 

Mammalian  type      ...         ...           ..          ...          ...  ..  ...  112 

Trypanosomes  ...         ...         ...         ...         ...         ...         ...  ...  ...  171 

Cultivation,  method  of    ...          ...         ...          ...         ...  ...  ...  152 

Examination  of  blood  for            ...          ...         ...         ...  ...  ...  114 

In  blood  plasma  ...          ...         ...         ...         ..           ...  ...  ...  no 

Ofbirds         ..           ...         ...         ...         ...          ...  ...  ...  1 10 

Offish            110 

(Mammalian)        ...         ...         ...         ...          ...         ...  ...  ...  111 

Types  of       1 1 1 

Multiplication  by  fission             ...         ...          ...         ...  ...  ...  115 

Relationship  of  spirochcetse  to    ...         ...         ...          ...  ...  ...  120 

Species  non-pathogenic  to  full-grown  rats       ...          ...  ...  ...  112 

Staining 114,  115 

With  carbol  fuchsin  ...  ...  ...         ...  ...  114,  115 

With  Leishman's  stain        ...         ...         ...          ...  ...  ...  115 

Trypanosomiasis          ...         ...         ...         ...         ...         ...  ...  ...  70 

See  also  Sleeping  sickness          ...         ...         ...         ...  ...  ...  113 

Auto-agglutination  of  blood-cells  in      ...          ...         ...  ...  ...  115 

Leucocyte  variation  in     ...  ...         ...         ...  ...  ...  57,  59 


index  533 

Trypanosomiasis-  -continued.  PAGE 

Mode  of  transmission       ...         ...  ...  ...  ...  ...  ..  116 

(Rhodesian),  obstinate  nature  of  ...  ...  ...  ...  ...  117 

Parasite  causing       ...          ...  ..  ...  ...  ...  ...  1 1 7 

Symptoms  of        ...         ...         ...  ...  ...  ...  ...  ...  113 

Tsetse-fly  disease,  see  Nagana 

Tubercle,  antiformin  method  of  examining  for  ...  ...  ...  ...  480 

Tubercle  bacilli            ...          ...         ...  ...  ...  ...  ...  ...  398 

Turkey  gnats    ...         ...         ...         ...  ...  ...  ...  ...  ...  I72 

Typhoid  bacilli,  harboured  by  Crustacea  ...  ...  ...  ...  ...  3°9 

Typhoid  fever  : — 

Diazo-reaction  of  urine  as  test  in  ...  ...  ...  ...  ...  377 

Germs  conveyed  by  diptera       ...  ...  ...  ...  ...  ...  160 

Leucocyte  variation  in    ...           ..  ...  ...  ...  ...  57,  59 


Uganda,  unnamed  species  of  piroplasma  found  in  monkey  in 

Ungues,  definition  of  ... 

Upas  tree  {see  Antiaris  toxic  aria). 

Uranotania 

Larvoe  of,  respiratory  syphon  in 
Urea,  diminution  of.  in  urine,  in  beri-beri  ... 
Urine  : — 

Bacteria  in 

Bile  in,  in  malaria 

Changes  in  beri-beri  cases 

Estimation  of  quinine  excreted  in 

Examination  of,  in  the  Tropics 

Filar  ia  ban  croft 'i  in 

Haemoglobinuric,  method  for  diagnosis 

Indican  in,  method  of  detection 

Medium  for  growth  of  organisms 

Rate  of  secretion  in  blackwater  fever  ... 

Solution  for  test  of  diazo  reaction  in    ... 
Urobilin,  discharge  of,  accompanies  pigment  deposits 

In  feces 

In  malaria 

Spectrum  of 


Vax  Gieson's  method  of  staining  

Description  of 
Verallina 

Vermipsylla  alakurt  (Flea),  mouth-piece  of 

Vertebrates,  cold-blooded,  blood  of,  hjemogregarines  common  in 
Vertex,  definition  of 
Vibrios 

Vincent  on  leucocyte  variation 
Viscera,  method  of  preserving  museum  specimens  of 


...    103 

...  165 

...   231 

...   271 

...  376 

•••     375 

•••     373 

■  •     376 

■•■     377 

•  ••     37i 

••■     372 

■••     374 

•••     374 

•••     375 

■■■     373 

■••     377 

315.  316 

333.  334 

333.  334 

•■•     334 

86,88 

...       86 

...     231 

...     284 

...     105 

...     162 

...     390 

...       57 

25,  26 

534  INDEX 

PAGE 

Wanki.yn's  process  of  estimating  free  ammonia  ...         ...         ...     430 

Ward,  II.  B.,  characteristics  of  canine  tapeworms  ...         ...         ...     351 

Wasp,  characters  of    ...  ...         ...  ...  ...  ...  ...  ...     157 

Water  :  — 

Bacteriological  examination  of ...  ...  ...  ...  ...        413,426 

Biological  examination  of  ...  ..         ...         ...         ...  ...     426 

Chemical  analysis  of       ...  ...         ...  ...         ...         ...         ...     426 

Chemical  substances  sought  for  in        ...         ...         ...         ...         ...     426 

Collection  for  examination         ...         ...         ...  ...         ...         ...     413 

(Distilled)  for  use  in  laboratory...         ...  ...         ...         ...         ...         4 

Examination  for  Koch's  comma  bacillus         ...         ...  ...         416,  417 

Of  plates  and  tubes  ...         ...         ...  ...  ...         ...     414 

Tubes  for  Bacillus  coli       ...         ...         ...  ...         ...     415 

Hardness  in  ...  ...  ...  ...  ...  ...  ...         434)435 

Inoculations  for  bacteriological  examination  of  ...  ...         ...     414 

Oxygenation,  for  breeding  larvae  of  mosquitoes         ...  ...         ...     274 

Physical  examination  of  ...  ...  ...  ...  ...  ...  ...     426 

Purity  of,  estimation        ...         ...  ...  ...         ...         ...        434,  435 

Water-butts  as  artificial  breeding-places  for  mosquito  larva?        ...         ...     278 

Water-plants,    stationary     and     floating,     favouring     development     of 

mosquito  larvre  ...         ...  ...  ...  ...         ...  ...     275 

Water-tank  for  laboratory      ...      .    ...  ...  ...  ...         ...         ...     2,3 

Water  :  — 

Deep  417 

Surface      ...  ...  ..  ...  ...  ...  ...         ...  ••■     4'7 

Temporary  and  permanent,  as  breeding-places  of  mosquito  larvae  ...     275 
Watson,  report  by,  as  regards  prophylaxis  in  malaria        ..  ...         470,  471 

Weather,  effect  upon  stains    ...  ...         ...         ...  ...         ...  ...       52 

Weber's  test  for  blood  in  feces         ..  ...         ...         ...         ...         ...     332 

Weight    of    organs   of    body,    variation    in    health    and    disease    from 

European  standard      ...  ...         ...         ...         ...  ...  ...       22 

Weil's  disease 373 

Whip-scorpions  296 

Whip-worms 328,  329 

Eggs  of 34i 

Preservation  and  examination  of  ...  ...  ...         ...  477-479 

Wright,  Sir  A.  E.,  F.R.S.,  discoverer  of  opsonins  152 

Wright's  glass  tubes  for  obtaining  and  diluting  blood  serum       ...  ...     14S 

India-rubber  teats  for  drawing  up  fluid  into  mixing  chamber  149,  150 

Method  of  estimating  coagulation  time  of  blood         ...  ...  ...      142 

Tubes  with  air  and  mixing  chambers,  estimation  of  isotonic  strength 

of  serum  by...         ...         ...  ...         ...         ...  ...      145 

Wyeornyia         ...         ...         ...  ...  ...  ■••         •••         •■•         •••     232 

Characters  of       ...         168 


Xylol  :  — 

Removal  of,  from  sections         ...  ...  ...  ...  ...         ...       39 

And  paraffin  method  of  imbedding       ...         ...         ...  ...         ...       32 


INDEX 

YAWS,  Spirochata  pertcmiis  present  in 
Yeasts    ... 

Spore-bearing  and  non-spore  bearing 
Yellow  fever,  fatty  degeneration  in  ... 

Transmission  by  mosquito  (Stegomyia  fasciaia) 

Value  of  charts  in 
Yellow  pigment,  chemistry  of 

Diseases  in  which  found... 

Distribution  of     ... 

Evidence  of  blood  destruction  .. 


535 

PAGE 
...  119 
390,  421 
421 
319 
259 
476 

313 
313 
3H 
314.  316 


Zenker's  fluid,  fixation  of  tissues  by 
Ziehl-Neelson's  method  of  staining 
Zinc  in  water,  test  for,  qualitative 

In  water,  test  for,  quantitative 
Zygotes... 

Development  of  ... 

Formation  of 

Proportion  of  gametocytes  forming 


29 

396 
428 
428 
253 
254 
109 

254 


C0||U|MMM.,1/.ERS,TVL,BRAR.ES 


0047960310 


COLUMBIA  UNIVERSITY  LIBRARY 

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expiration  of  a  definite  period  after  the  date  of  borrowing, 
as  provided  by  the  rules  of  the  Library  or  by  special  ar- 
rangement with  the  Librarian  in  charge. 


DATE  BORROWED 

DATE  DUE 

DATE  BORROWED 

DATE  DUE 

AUG  1  4  19/ 

C2S(239)M100 

